Method for detecting allergen in drug-induced allergic shock

Abstract

The invention belongs to the field of forensic detection, and particularly relates to a method for detecting allergens in drug-induced allergic shock. Based on a type I hypersensitivity reaction mechanism, carboxylated microspheres are used for cross-linking an anti-IgE antibody, an IgE-allergen compound in a sensitization stage of a detection sample is combined and captured, interference of residual components of the sample is removed through multiple times of cleaning, the antibody and drug binding protein are digested by enzyme to dissociate drug components, and the immunogenicity of the IgE-allergen compound in the sensitization stage of the detection sample is improved. And the LC-MS is used for qualitatively detecting medicine components so as to achieve the purpose of detecting the medicine allergen.

Description

Technical Field

[0001] This invention belongs to the field of forensic testing, specifically relating to a method for detecting allergens in drug-induced anaphylactic shock. Background Technology

[0002] Anaphylactic shock is a severe, systemic, and life-threatening systemic reaction, primarily a type I hypersensitivity reaction mediated by IgE. When the body is first exposed to an allergen, plasma cells produce large amounts of IgE, which binds to mast cells via the high-affinity IgE receptor FcεR1 on the mast cell membrane. Upon re-exposure, multiple IgE molecules bind to a single antigen, bridging the antigen and activating mast cells. Mast cells then degranulate and release a series of active mediators, playing a crucial role in the development and progression of anaphylactic shock. Therefore, in the medical field, IgE is key to identifying whether a reaction is allergic.

[0003] However, certain parasitic diseases, fungal infections, Staphylococcus aureus infections, postmortem hemolysis, and coagulation can all affect the results of serum total IgE measurements. Furthermore, whether the total IgE level rises during an allergic reaction is also related to the nature of the antigen and genetic factors. Therefore, relying solely on serum total IgE testing to diagnose anaphylactic shock is unreliable. Detecting allergen-specific IgE to identify the allergen is a more reliable method that provides more supplementary information.

[0004] Anaphylactic shock, lacking specific pathological morphological changes, is one of the challenges in forensic pathology identification. Those who die from anaphylactic shock typically develop symptoms within 30 minutes of exposure to an allergen. Due to the sudden onset, it is difficult to detect clear morphological changes in the deceased; autopsies often only reveal general signs such as congestion and edema of organs and tissues, indicating sudden death. Because anaphylactic shock often does not exhibit specific pathological morphological changes, diagnosis requires consideration of allergy and medication history, and the exclusion of other causes of death such as asphyxiation and poisoning, thus posing significant difficulties for forensic identification.

[0005] Currently, in my country, corpses are primarily preserved by freezing before autopsy. Freezing effectively slows down the decomposition process and extends preservation time. However, freezing can also cause non-specific postmortem changes, such as fractures and suture separation in weak areas of the skull base, ice crystals within the body, ice crystal cracks in pathological sections, and severe hemolysis caused by the rupture of red blood cells throughout the body after freeze-thaw cycles. Clinical studies have shown that hemolysis severely interferes with the detection of multiple clinical indicators. This is due to factors including interference from the concentration difference of substances inside and outside ruptured blood cells, as well as interference from hemolysis-based colorimetric and immunoassay methods. In forensic practice, hemolysis of samples is more severe than in clinical settings, and ideal samples cannot be obtained through re-collection of blood, exhibiting irreversible characteristics. This presents another challenge for postmortem allergen detection.

[0006] Multiple forensic retrospective analyses have shown that drugs are the primary cause of anaphylactic shock deaths in my country. The detection of allergen-specific IgE in post-mortem blood and pericardial fluid is the gold standard for drug-induced anaphylactic shock. Accurate identification of the allergenic drug can not only effectively diagnose anaphylactic shock but also determine the causative drug, providing scientific and direct evidence for potential medical disputes and possessing extremely high forensic value.

[0007] Currently, clinical testing primarily targets common allergens in daily life, such as food (milk, nuts), dust mites, and pet dander. There is no definitively proven and universally effective method for detecting allergens in drug-induced anaphylactic shock. Studies have shown that drugs, after entering the body, typically act as haptens, requiring binding to proteins in the body to exert their full antigenicity. This stimulates the body to produce specific IgE, which then binds to the drug, triggering a subsequent hypersensitivity reaction.

[0008] Regarding the detection of drug allergens, current forensic literature only mentions the application of Immunocap reagents for screening a limited number of drug allergens. Unfortunately, this reagent and related Phadia instruments are not available for sale in my country. https: / / www.thermofisher.cn / order / catalog / product / 12390001 The webpage indicates that Phadia devices are not available for sale in China. Figure 1 Therefore, the detection technology for drug allergens in cases of drug-induced anaphylactic shock and death is currently a technological gap in China. Summary of the Invention

[0009] IgE-mediated type I hypersensitivity is the classic mechanism of anaphylactic shock. Drug-induced anaphylaxis (DIA) is a common type I hypersensitivity reaction, mediated by specific IgE induced by the sensitizer. During the sensitization phase, the drug is usually a hapten; after entering the body, it binds to human proteins to become a complete antigen, inducing allergen-specific B cells to produce IgE. The Fc fragment of IgE binds to the FcεRI high-affinity receptor on the surface of mast cells or basophils, forming sensitized mast cells or basophils, thus sensitizing the body to the allergen. During the sensitization phase, when the allergen re-enters the sensitized body, it binds to IgE on the surface of sensitized mast cells or basophils. When two or more IgE cells simultaneously bind to the allergen and form cross-links, an activation signal is initiated, leading to degranulation and the release of various intracellular bioactive mediators such as histamine, proteases, and prostaglandins. This results in phenomena such as increased vascular permeability, smooth muscle contraction, increased glandular secretion, and eosinophil activation.

[0010] This invention is based on the aforementioned type I hypersensitivity reaction mechanism. It uses carboxylated microspheres cross-linked with anti-IgE antibodies to bind and capture IgE-allergen complexes in the sensitization stage of blood and pericardial fluid. After multiple washes to remove interference from residual blood components, the antibody and drug-binding protein are digested with enzymes to release the drug components. LC-MS is then used for qualitative detection of the drug components to achieve the purpose of detecting drug allergens. Figure 2 ).

[0011] On one hand, the present invention provides a method for detecting allergens in drug-induced anaphylactic shock, comprising: S1. The sample to be tested is contacted with a capture reagent conjugated with anti-IgE antibody to obtain an incubation mixture; S2. Add protease to the incubation mixture obtained in step S1 for enzymatic digestion, and collect the supernatant; S3. Perform LC-MS analysis on the supernatant obtained in step S2, and obtain the allergens in drug-induced anaphylactic shock through the LC-MS results; The samples to be tested are: hemolyzed blood from the corpse and / or pericardial fluid from the corpse.

[0012] In some embodiments, in step S1, the capturing agent includes at least one of latex microspheres and magnetic microspheres; in some embodiments, the surface of the capturing agent contains carboxyl groups.

[0013] In some implementations, step S1 includes: After contacting the sample to be tested with the capture reagent conjugated with anti-IgE antibody, it was incubated at 37°C for 2-5 hours. After incubation, the supernatant was discarded by centrifugation to obtain the incubation mixture.

[0014] In some embodiments, the first centrifugation is performed at 10,000-15,000 r for 5-10 min; in some embodiments, after discarding the supernatant after the first centrifugation, a washing step is further included; in some embodiments, the washing step includes: adding PBS and sonicating, followed by a second centrifugation; in some embodiments, the second centrifugation is performed at 10,000-15,000 r for 5-10 min; in some embodiments, the washing is repeated 2-8 times.

[0015] In some embodiments, the protease in step S2 is papain; in some embodiments, the enzymatic hydrolysis conditions are incubation at 37°C for 1-5 hours; in some embodiments, after enzymatic hydrolysis and incubation, deionized water is added, and the supernatant is collected after a third centrifugation; in some embodiments, the conditions for the third centrifugation are 10000-15000 r for 5-10 min.

[0016] In some embodiments, step S3 includes a pretreatment step before performing LC-MS detection on the supernatant obtained in step S2. The pretreatment step includes mixing the supernatant with a methanol-acetonitrile mixture, centrifuging for the fourth time, and retaining the obtained supernatant.

[0017] In some embodiments, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile mixture is (1.5-2.5):(0.5-1.5); in some embodiments, the conditions for the fourth centrifugation are 11000-16000 r for 2-8 min.

[0018] In some embodiments, in step S3, the liquid chromatography conditions are at least one of the following a1-a4: a1. Column type: Any one of the following: C4, C8, C12, and C18 column types; a2. Mobile phase: 15-30 mmol ammonium acetate and 0.05-0.2% formic acid buffer: acetonitrile; a3. Flow rate: 0.1-0.5 ml / min; a4. Injection volume: 2-10 µL.

[0019] In some implementations, in step S3, the mass spectrometry conditions are at least one of the following b1-b4: b1. ESI ion source; b2. Drying airflow rate: 5-20 L / min; b3. Atomizer pressure 40-60 psi; b4. Multiple Response Monitoring (MRM) mode.

[0020] In some embodiments, the drug includes at least one of cefoperazone, sulbactam, levofloxacin, clindamycin, creatine phosphate sodium, ribavirin, ceftriaxone, cimetidine, aztreonam, gentamicin, ambroxol, moxifloxacin, dexamethasone, and pazufloxacin.

[0021] On the one hand, the present invention provides an application of the method thereof, which is at least one of the following c1-c2: c1. Detecting allergens in the sample that bind to specific IgE; c2. Sensitizing drugs in subjects who have experienced anaphylactic shock due to forensic drug testing.

[0022] On one hand, the present invention provides a system for detecting allergens in drug-induced anaphylactic shock, comprising: 1) Inspecting components; 2) Data processing components; 3) Output components; The detection component is configured to: contact the sample to be tested with a capture reagent conjugated with anti-IgE antibody to obtain an incubation mixture, add protease to the incubation mixture, take the supernatant after enzymatic digestion, and perform LC-MS detection on the supernatant; The data processing component is configured to: a. receive detection data of the sample to be tested; b. store the detection data of the sample to be tested; c. compare the sample to be tested and the drug standard with: i. retention time, ii. qualitative ion and iii. ion pair abundance ratio; d. if all three indicators i-iii are consistent with the drug standard, then the sensitizer of the sample to be tested is the drug corresponding to the drug standard. The result output component is configured to output the allergens of the sample to be tested; The samples to be tested are hemolyzed blood from corpses and / or pericardial fluid from corpses.

[0023] The advantages of this method are twofold. First, it enables systematic detection of multiple, even common, drug allergens, overcoming the limitations of the "one drug, one method" approach. Second, by qualitatively detecting the antigen-IgE complex to identify allergen-specific IgE, its diagnostic value surpasses that of simply screening for specific IgE. This is because the antigen-IgE complex is formed only during the sensitization phase; therefore, the allergen detected by this method is the one that triggered the current allergic reaction. Even if the body is sensitive to other allergens, they cannot be detected in the non-sensitized state because the antigen-IgE complex has not formed. This effectively eliminates interference from other non-sensitizing allergens, enhancing its diagnostic value and practicality. Attached Figure Description

[0024] Figure 1 for https: / / www.thermofisher.cn / order / catalog / product / 12390001A screenshot of the webpage.

[0025] Figure 2 This is a schematic diagram of the allergen-specific IgE detection method based on the type I hypersensitivity reaction mechanism of the present invention.

[0026] Figure 3 A schematic diagram of the process for detecting allergen-specific IgE by cross-linking antibodies with carboxyl-modified latex microspheres using LC-MS.

[0027] Figure 4 The image shows a photograph of the carboxyl-modified latex microspheres used in the examples.

[0028] Figure 5 The images show the staining of mast cells and eosinophils in Example 1, where: A: Mast cell staining in lung tissue of allergic rats, 400X field of view; B: Mast cell staining in lung tissue of control rats, 400X field of view; C: Eosinophil staining in lung tissue of allergic rats, 400X field of view; D: Eosinophil staining in lung tissue of control rats, 400X field of view.

[0029] Figure 6 The images show the MRM chromatograms of the autopsy blood samples and standards in Group ① of Example 2, where: S1: Cefoperazone standard detection chromatogram; DIA-1: Cefoperazone detection chromatogram of DIA-1 sample; S2: Clindamycin standard detection chromatogram; DIA-2: Clindamycin detection chromatogram of DIA-2 sample.

[0030] Figure 7 The images show the MRM chromatograms of the autopsy blood samples and standards in Group ① of Example 2, where: S3: chromatogram of levofloxacin standard; DIA-3: chromatogram of levofloxacin in DIA-3 sample; S4: chromatogram of ribavirin standard; DIA-4: chromatogram of ribavirin in DIA-4 sample.

[0031] Figure 8 The images show the MRM chromatograms of the autopsy blood samples and standards in Group ① of Example 2, where: S5: Cimetidine standard detection chromatogram; DIA-5: Cimetidine detection chromatogram of DIA-5 sample; S6: Ambroxol standard detection chromatogram; DIA-6: Ambroxol detection chromatogram of DIA-6 sample.

[0032] Figure 9 The image shows the MRM chromatogram of levofloxacin detected in the pericardial fluid sample of DIA-3 from the autopsy in Group ① of Example 3.

[0033] Figure 10 The image shows the MRM chromatogram of cimetidine in the pericardial fluid sample of DIA-5 from the autopsy in Group ① of Example 3. Detailed Implementation

[0034] The above embodiments are merely preferred embodiments of this application and should not be construed as limiting the scope of protection of this application. Any non-substantial changes and substitutions made by those skilled in the art based on this application shall fall within the scope of protection claimed by this application.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] The reagent information involved in the examples is shown in Table 1.

[0037] Table 1

[0038] Example 1 Penicillin allergy is the most common type of anaphylactic shock caused by drugs. Penicillin itself is not immunogenic; however, its degradation products and endogenous or exogenous impurities generated during synthesis, transportation, storage, or use can covalently bind to proteins in the body, stimulating the production of specific IgE, thus sensitizing the body. Re-exposure can trigger an allergic reaction, and in severe cases, lead to anaphylactic shock and death. Penicillin thiazolic acid is one such sensitizing agent. Studies have shown that penicillin thiazolic acid, the ring-opening product of the β-lactam ring of penicillin, can bind to the tissue compatibility complex (MHC) on the surface of T cells or to polypeptides embedded in the cell membrane. The resulting complex stimulates T cells, thereby causing an allergic reaction. In this example, the rat penicillin thiazolic acid allergy model uses penicillin thiazolic acid bound to bovine serum albumin (BSA) as the sensitizer, effectively simulating the process by which the drug enters the human body as a hapten and binds to human proteins to form a complete antigen.

[0039] Materials and Methods 1. Establishment of a rat model of penicillin-thiazole protein allergy 1.1 Animal Grouping A total of 20 SPF-grade male 8-week-old SD rats, weighing 300-320 g, were provided by Beijing Huafukang Biotechnology Co., Ltd., with 10 rats serving as the allergy group and 10 rats serving as the control group.

[0040] 1.2 Preparation of Penicillin Thiazole Protein Weigh 0.96 g of penicillin G sodium (at 1 g, the final injection concentration is 2 mg / ml), add 10 mL of 0.20 mol / L NaOH solution to adjust the pH to >12, stir magnetically at 27℃ for 30 min, then adjust the pH to 8.7 with 1 mol / L hydrochloric acid, and incubate at 37℃ for 48 hours to obtain a penicillin-thiazolic acid solution. Then weigh 0.9 g of bovine serum albumin (BSA), dissolve it in 20 mL of physiological saline, add it to the prepared penicillin-thiazolic acid solution, and add physiological saline to bring the volume to 100 mL. Mix thoroughly, adjust the pH to 7.5, and refrigerate at 4℃. Take 10 mL of the above penicillin-thiazolic acid solution and dilute it 5 times with physiological saline as the sensitization and challenge solution.

[0041] 1.3 Dosing regimens for the allergy group and the control group Each rat was intraperitoneally injected with 0.5 ml of the above penicillin-thiazole protein solution on days 1, 3, and 5. Fourteen days after the last sensitization, each test animal was challenged by intravenous injection of 0.5 ml of the corresponding test substance (at a concentration 1 times that of the sensitization), and the symptoms of allergic reaction were recorded.

[0042] In the control group, physiological saline was used instead of saline for sensitization and challenge. Animals in both the sensitized and control groups were euthanized by CO2 asphyxiation.

[0043] 1.4 Animal-based materials Blood was collected immediately after animal euthanasia. Five animals from each of the allergy group and control group had their blood drawn, and the serum was immediately separated by centrifugation at 3000 g for 15 min. The remaining five animals from each group had 1.5 ml of blood collected and subjected to a freeze-thaw cycle of -20°C followed by room temperature freezing five times to lyse the blood. The remaining blood was then immediately centrifuged at 3000 g for 15 min to separate the serum. All blood samples and the lysed blood were stored at -80°C for testing. The serum and hemolyzed blood were divided into 3 groups: A. Serum and hemolyzed blood of animals in the penicillin-thiazole protein allergy group.

[0044] B. False Positive Group: Serum and hemolyzed blood from control group animals were collected. The circulating blood volume of rats was calculated based on literature reports (average 64 ml / kg). The addition ratio was calculated based on the last tail vein injection of 0.5 ml of challenge solution. For example, a rat weighing 400 g would have a circulating blood volume of approximately 25.6 ml, requiring a ratio of 980.5 μl of control group animal serum or hemolyzed blood + 19.5 μl of challenge solution.

[0045] C. Control group: Serum and hemolyzed blood of animals in the control group.

[0046] Lungs were harvested from animals in the allergy group and the control group, washed with physiological saline, and then fixed in 4% paraformaldehyde solution.

[0047] 2. ELISA method for detecting serum IgE and trypsin-like proteins. The concentrations of IgE and trypsin in the serum of the allergic group and the control group were detected using a rat IgE detection kit (CSB-E07984r, Wuhan Huamei) and a rat trypsin detection kit (CSB-E13627r, Wuhan Huamei). The operation procedure was performed according to the kit instructions. Standards and serum samples were added sequentially to the wells of a 96-well plate coated with anti-IgE antibody and anti-trypsin antibody, and incubated at 37°C for 1 h. After discarding the liquid, biotin-labeled antibody working solution was added, and the plate was incubated at 37°C for 1 h. The liquid in the wells was then discarded and the plate was washed. Horseradish peroxidase-labeled avidin working solution was added, and the plate was incubated at 37°C for 1 h. After thorough washing, the plate was developed with chromogenic reagent. The absorbance (OD value) was measured at 450 nm using a microplate reader (SYNERGY H1, BioTek) within 5 min, and the sample concentration was calculated.

[0048] 3. Histopathological staining Lungs fixed in 4% paraformaldehyde solution were embedded in paraffin and sectioned. Mast cells in the lung paraffin sections were stained and observed using mast cell staining solution (toluidine blue method), and eosinophils in the lung paraffin sections were stained and observed using eosinophil staining kit (Sirius red method).

[0049] 4. Detection of drug antigens that bind specifically to penicillin-thiazole protein IgE. In this embodiment, the detection of drug antigens specifically bound to IgE in serum and hemolyzed blood is performed through four steps: cross-linking anti-IgE antibodies with carboxyl-modified latex microspheres, capturing IgE in blood samples with cross-linked antibody microspheres, decomposing antibodies with papain, eluting penicillin thiazole protein antigen, and mass spectrometry detection. A flowchart is shown below. Figure 3 .

[0050] 4.1 Carboxyl-modified latex microspheres crosslinked with anti-IgE antibodies 4.1.1 Reagent preparation: 1) Activation / coupling buffer: 50 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 6.0 (adjust pH with NaOH solution).

[0051] 2) Blocking solution: 50 mM Tris, pH 8.0, 0.5% (w / v) casein (adjust pH with 4M HCl).

[0052] 3) 200 mM EDC solution: 19.2 mg EDC·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) was added to 500 μl of pure water.

[0053] 4) 200 mM Sulfo-NHS (N-hydroxythiosuccinimide): 21.7 mg Sulfo-NHS was added to 500 μl of activation / coupling buffer.

[0054] 4.1.2 Operating Procedures ① Microsphere washing and activation 1) Add 125 μl of blue carboxyl-modified latex microspheres (4% solids content) (ABS9710), i.e., 5 mg of microspheres, to a centrifuge tube, add 1 ml of activation / coupling buffer and mix thoroughly. Centrifuge at 12000 rpm for 7 minutes and discard the supernatant.

[0055] 2) Repeat step 1 twice. Note that after adding the activation / coupling buffer, use vortexing and sonication to resuspend the microspheres in the liquid. After the final centrifugation, resuspend the microspheres in 1 ml of activation / coupling buffer.

[0056] 3) Quickly add 12 µl of 200 mM EDC and 120 µl of 200 mM Sulfo-NHS to 1 ml of washed microspheres (from step 2) and 1 ml of activation / coupling buffer for resuspension. Vortex to mix and then incubate at room temperature for 30 minutes.

[0057] 4) After incubation, centrifuge at 12000 rpm for 7 minutes and discard the supernatant. Add 1 ml of activation / coupling buffer to wash the microspheres and mix thoroughly. Centrifuge at 12000 rpm for 7 minutes.

[0058] 5) Repeat step 4 twice. Resuspend the microspheres in 850 µl of activation / coupling buffer and vortex mix with sonication to ensure the microspheres are in a monodisperse suspension.

[0059] ②Antibody conjugation 1) Based on the target preparation concentration of 20 mg anti-rat IgE antibody per gram of microspheres, 100 µl of anti-rat IgE antibody (Abcam, ab11666) (concentration of 2 mg / ml) was added to 900 µL of microsphere suspension. The mixture was thoroughly mixed. The total liquid volume was now 1 ml. The suspension was incubated on a rotating plate at room temperature for 2.5 hours.

[0060] 2) After incubation, add 15 µl of ethanolamine to 1 ml of microspheres (in a fume hood). Vortex and mix on a rotary wheel for 30 minutes to quench excess carboxyl groups on the microspheres. After quenching, centrifuge at 12000 rpm for 7 minutes and discard the supernatant.

[0061] 3) Add 1 ml of blocking buffer, vortex and sonicate to suspend the microspheres, mix on a rotary wheel at room temperature for 2 hours, centrifuge at 12000 r for 7 minutes, discard the supernatant, add 1 ml of blocking buffer, vortex and sonicate to suspend the microspheres, and store at 4℃ for later use.

[0062] 4.2 Cross-linked antibody microspheres capture IgE in blood samples Take 1 ml of serum and 1 ml of hemolyzed blood from the allergy group, false positive group, and control group. Add 50 µl of cross-linked anti-rat IgE antibody microspheres to each microsphere and incubate at 37°C for 3 hours, vortexing every half hour. After incubation, centrifuge at 12000 rpm for 7 minutes, discard the supernatant, add 1 ml of PBS, vortex to mix, and sonicate to suspend the microspheres. Centrifuge at 12000 rpm for 7 minutes. Repeat the washing process 5 times to remove residual penicillin-thiazole protein components from the serum.

[0063] 4.3 Papain breaks down antibodies and washes away penicillin-thiazolic acid allergens. After the final centrifugation and discarding of the supernatant, add 200 µl of 20 mg / ml papain solution to the centrifuge tube, vortex to mix, and then sonicate to suspend the microspheres. Incubate at 37°C for 2 hours, vortexing every 30 minutes. After incubation, centrifuge at 12000 rpm for 7 minutes, and retain the supernatant (approximately 130 µl) for testing. The samples to be tested are numbered 1-6, as detailed in Table 2.

[0064] Table 2 Sample Numbers to be Detected by Mass Spectrometry

[0065] 5. LC-MS detection Add 400 µl of methanol-acetonitrile mixture (methanol:acetonitrile = 2:1, v / v) to the supernatant samples after treatments 1-6. Vortex for 3 min to mix thoroughly, centrifuge at 13000 r for 5 min, and retain the supernatant for testing.

[0066] This experiment used an Agilent 1260-6420 LC-MS / MS system: the column used was an Agilent Zorbax SB C18 (2.1 mm × 50 mm, 1.8 μm); column temperature: 30℃; mobile phase: 20 mmol / L ammonium acetate and 0.1% formic acid buffer: acetonitrile; flow rate: 0.30 mL / min; elution program is shown in Table 3. Table 3. Penicillin-thiazolic acid elution procedure

[0067] Injection volume 5 µL; ESI ion source, positive ion mode; drying gas flow rate 10 L / min; nebulizer pressure 50 psi; multiple reaction monitoring (MRM) mode, detection conditions are shown in Table 4 below.

[0068] Table 4

[0069] 6. Statistical Analysis Experimental data were processed using SPSS 20.0. Serum IgE and trypsin levels were analyzed. t The tests were compared and analyzed.

[0070] result 1. Evaluation of animal models of penicillin-thiazole protein allergy 1.1 Observation of allergic reactions in animals After the allergic rats were injected with the provocation solution via the tail vein, they exhibited symptoms such as increased activity, scratching around the mouth, lying down, and rapid breathing; some rats also experienced convulsions. No abnormal activity was observed in the control group rats.

[0071] 1.2 Serum IgE and trypsin levels Serum IgE and trypsin levels in the allergy group and control group were detected using an ELISA kit. The results showed that the serum IgE and trypsin levels in the allergy group were higher than those in the control group, and the differences were statistically significant (P<0.05, Table 5).

[0072] Table 5. Serum IgE and trypsin levels in rats (n=10)

[0073] The data in the table are presented in the form of "mean ± standard deviation"; "This indicates that the difference was statistically significant compared with the control group (P<0.05).

[0074] 1.3 Staining of mast cells and eosinophils Mast cell staining solution and eosinophil staining kit were used to stain mast cells and eosinophils in rat lung tissue sections. The results showed an increase in eosinophils and significant mast cell degranulation in the lung tissue of the allergic group rats, while no abnormalities were observed in the control group. Figure 5 ).

[0075] After sensitization, animal behavior, blood IgE, trypsin detection, and lung mast cell and eosinophil staining were observed. The allergic group all exhibited typical allergic reaction manifestations, confirming the successful establishment of an allergic animal model.

[0076] 2. Detection of drug antigens bound to penicillin-thiazole protein-specific IgE. LC-MS analysis revealed that penicillin-thiazolic acid was detected in samples 1 and 2 out of the six groups (1-6). The retention times and qualitative ion pairs of the target analyte in MRM mode were consistent with the standard. No penicillin-thiazolic acid was detected in the remaining samples (3, 4, 5, and 6).

[0077] result In this embodiment, a method using anti-IgE antibody-crosslinked microspheres to capture immune complexes and mass spectrometry to detect drug groups was designed to qualitatively detect specific IgE in penicillin-thiazol protein-allergic rats. The results showed that mass spectrometry detection only detected penicillin-thiazol acid in the serum of allergic animals and in completely hemolyzed samples. Although penicillin-thiazol protein solution was artificially added to the serum of the control group, it was still not detected by mass spectrometry, and it was also not detected in the control group. This confirmed that the penicillin-thiazol acid detected in the allergic group was not a free residue in the blood or a certain component in the serum, but rather a penicillin-thiazol acid structure carried by the allergen in the antibody-captured immune complex, i.e., the penicillin-thiazol acid structure in penicillin-thiazol protein. This indicates that the established method of anti-IgE antibody-crosslinked microspheres to capture immune complexes and mass spectrometry to detect drug groups successfully qualitatively detected the drug antigen bound to specific IgE in penicillin-thiazol protein-allergic rats.

[0078] Example 2 1. Blood sample collection during autopsy This study collected blood samples from 18 autopsies conducted at the China Medical University Forensic Science Center between 2023 and 2025. All blood samples were extracted from the left ventricle via a sterile Pasteur pipette incision at the root of the aorta. The cause of death in all cases was determined through systematic autopsy, toxicology, and postmortem biochemical analysis. Six cases were due to drug-induced anaphylaxis (DIA); six cases were non-allergic deaths with a history of drug exposure (Medication Exposure, ME), and the exposed drugs covered suspected allergens in the DIA group; and six cases were non-allergic deaths without a history of drug exposure. Detailed information is shown in Table 6.

[0079] Table 6 Information on 18 Included Cases

[0080] The above 18 samples were divided into 4 experimental groups, namely: ①: Allergy group, blood samples from 6 cases of anaphylactic shock death; ②: Drug exposure history group, 6 cases of non-allergic deaths had a history of drug exposure in their blood; ③: False positive group: Blood samples from 6 cases of non-allergic deaths with no history of drug exposure were used. Standards were applied to the 6 cases of anaphylactic shock deaths, and a false positive control with a drug concentration of 10 μg / ml was prepared to ensure that the drug concentration in the false positive group was higher than that in the allergy group. ④: Control group, 6 cases of non-allergic deaths with no history of drug exposure. 2. Carboxyl-modified latex microspheres cross-linked with antibodies for LC-MS detection of allergen-specific IgE binding sensitizers. In this embodiment, the detection of allergens specifically bound to IgE in hemolyzed blood involves four steps: cross-linking anti-IgE antibodies with carboxyl-modified latex microspheres, capturing IgE in the blood sample with cross-linked antibody microspheres, decomposing antibodies with papain, eluting the allergen drug components, and mass spectrometry detection. A flowchart is shown below. Figure 3 .

[0081] 2.1 Carboxyl-modified latex microspheres crosslinked with anti-IgE antibodies The steps are the same as in section 4.1 "Carboxyl-modified latex microspheres crosslinked with anti-IgE antibody" in Example 1. Only step 1 in antibody conjugation (②) differs slightly in this example: ②Antibody conjugation 1) Based on the preparation target of 20 mg anti-rat IgE antibody coating per gram of microspheres, 400 µl of anti-human IgE antibody (Abcam, ab99804) (concentration of 2 mg / ml) was added to 600 µL of microsphere suspension. The mixture was thoroughly mixed. The total liquid volume was now 1 ml. The suspension was incubated on a rotating plate at room temperature for 2.5 hours.

[0082] 2.2 Cross-linked antibody microspheres capture IgE in blood samples Take 1 ml of blood samples from each of the four autopsy groups (①, ②, ③, and ④), add 50 µl of cross-linked antibody microspheres (prepared in step "② Antibody Conjugation"), and incubate at 37°C for 3 hours, vortexing once every half hour. After incubation, centrifuge at 12000 rpm for 7 minutes, discard the supernatant, add 1 ml of PBS, vortex to mix, and sonicate to suspend the microspheres. Centrifuge at 12000 rpm for 7 minutes, repeating the washing operation 5 times to remove residual drug components in the blood.

[0083] 2.3 Papain breaks down antibodies and washes away drug components from allergens. After the final centrifugation and discarding of the supernatant, add 100 µl of 20 mg / ml papain solution to the centrifuge tube, vortex to mix, and then sonicate to suspend the microspheres. Incubate at 37°C for 2 hours, vortexing every 30 minutes. After incubation, add 50 µl of deionized water, centrifuge at 12000 rpm for 7 minutes, and retain the supernatant for testing.

[0084] 2.4 LC-MS detection After enzymatic digestion of the autopsy blood sample, the supernatant sample (approximately 130 µl) was added to 400 µl of methanol-acetonitrile mixture (methanol:acetonitrile = 2:1, v / v), vortexed for 3 min to mix thoroughly, centrifuged at 13000 r for 5 min, and the supernatant was retained for testing.

[0085] This experiment used an Agilent 1260-6420 LC-MS / MS system: the column used was an Agilent Zorbax SB C18 (2.1 mm × 50 mm, 1.8 μm); column temperature: 30℃; mobile phase: 20 mmol / L ammonium acetate and 0.1% formic acid buffer: acetonitrile; flow rate: 0.30 mL / min. The positive ion mode elution programs for 11 drugs—cefotaxime, gentamicin, clindamycin, moxifloxacin, dexamethasone, ambroxol, levofloxacin, cimetidine, ribavirin, creatine phosphate sodium, and ceftriaxone—are shown in Table 7.

[0086] Table 7. Positive ion mode elution procedures for 12 drugs

[0087] The elution procedures for aztreonam and sulbactam in negative ion mode are shown in Table 8.

[0088] Table 8. Negative ion mode elution procedure for aztreonam and sulbactam.

[0089] Injection volume 5 µL; ESI ion source, positive / negative ion mode; drying gas flow rate 10 L / min; nebulizer pressure 50 psi; multiple reaction monitoring (MRM) mode. Table 9 shows the detection conditions and limits of detection for suspected sensitizing drugs in autopsy blood samples from patients with drug-induced anaphylactic shock.

[0090] Table 9 LC-MS Detection Conditions for Sensitizing Drugs

[0091] result 1. Autopsy results of blood allergen testing in drug-induced anaphylactic shock LC-MS analysis revealed that all autopsy blood samples (group ①) contained positive drug samples. The retention times in MRM mode were consistent with the standards, the qualitative ion pairs of the target analytes were identical to the standards, and the ion pair abundance ratios also met the requirements of SF / T 0175—2024 "Liquid Chromatography-Mass Spectrometry Methods for the Examination of 238 Toxic (Drug) Substances in Blood and Urine" (Table 10). Figure 6-8 The remaining groups ②, ③, and ④ did not show corresponding chromatographic peaks at their respective retention times, indicating that no drugs were detected in them.

[0092] Table 10 Calculation results of drug detection and ion pair abundance ratio in autopsy blood samples of group ①

[0093] In this embodiment, anti-IgE antibody cross-linked microspheres were used to capture immune complexes, and LC-MS was used to detect drug groups. The drug-specific IgE binding of autopsy blood sensitized by drug-induced anaphylactic shock was qualitatively detected. The results showed that the drug component was only detected in the allergy group, and no drug component was detected in the drug exposure history group, false positive group, and control group.

[0094] Even when the false positive group prepared samples with drug concentrations far higher than those in the allergy group using standard reagents, the test results were still negative. Furthermore, the drug component detected in the allergy group was not the one with the highest concentration in its blood. This indicates that the drug component detected in the allergy group was not a free residue in the blood or a certain component in the serum, but rather a component carried by the allergen in the immune complex captured by the antibody. This is the sensitizing drug component that constitutes the antigen-induced anaphylactic shock.

[0095] This indicates that the method established in this invention, which involves capturing immune complexes with anti-IgE antibody cross-linked microspheres and detecting drug groups using LC-MS, successfully qualitatively detects drug allergen-specific IgE in the blood, providing a reliable method for the accurate identification of allergens in forensic drug-induced anaphylactic shock cases.

[0096] Example 3 In this embodiment, pericardial fluid from DIA-3 and DIA-5, cases of drug-induced anaphylactic shock (DIA) death in Example 2, was used as the test sample to verify the detection effect of the described detection method on pericardial fluid samples.

[0097] Except for the sample to be tested, the reagents, method steps and other parameters in this embodiment are the same as those in Example 2.

[0098] The results showed that both blood and pericardial fluid samples from the two cases tested positive for drugs, and the drug detection results in the pericardial fluid were consistent with those in the blood. Specifically: levofloxacin was detected in the pericardial fluid of DIA-3, with an ion-pair abundance ratio of 77.3%; cimetidine was detected in DIA-5, with an ion-pair abundance ratio of 59.3%, both meeting the requirements of SF / T 0175—2024 "Liquid Chromatography-Mass Spectrometry Methods for the Examination of 238 Toxic (Drug) Substances in Blood and Urine" (Table 11). Figure 9-10 ).

[0099] Table 11 Calculation results of drug detection and ion pair abundance ratio in pericardial fluid samples from group ① autopsy

[0100] Comparative Example Hemolyzed samples were detected by ELISA using a rat ovalbumin-specific IgE (OVA sIgE) enzyme-linked immunosorbent assay kit (CSB-E08913r).

[0101] I. Experimental Methods and Grouping: 1. Preparation of hemoglobin solution: Five ml of blood was drawn from the volunteer into an anticoagulant tube, centrifuged at 3000 g for 15 min, and the supernatant serum was removed. Physiological saline was added to 5 ml, and the washing was repeated 5 times to remove residual serum components. After the last centrifugation, the supernatant liquid was removed, and the lower layer of blood cells was frozen at -20°C and then slowly frozen at room temperature. This process was repeated 3 times to rupture all blood cells. The hemoglobin concentration was detected using Venzie's solution (cyanide methemoglobin method, Zhongshan Institute of Tianjin Modern High-Tech Research Institute) on a UV-1800 ultraviolet-spectrum spectrophotometer (Shimadzu Corporation, Japan). Physiological saline was then added to adjust the hemoglobin concentration to 200 g / L.

[0102] 2. Preparation of Gradient Hemolyzed Samples Prepare hemolyzed samples with hemoglobin concentrations of 20 g / L, 50 g / L, 100 g / L, and 150 g / L by mixing the hemoglobin solution with the sample diluent in the ELISA kit. Since no positive control was added, the actual results should all be negative.

[0103] 3. ELISA testing Following the operating procedures described in the instructions for the rat ovalbumin-specific IgE (OVA sIgE) enzyme-linked immunosorbent assay kit, negative control, positive control, and blank control wells were set up, and hemolysis-negative samples at four concentration gradients of 20 g / L, 50 g / L, 100 g / L, and 150 g / L were tested. All samples were diluted 1:200 before loading, as described in the instructions.

[0104] II. Results Visual inspection revealed significant discoloration in the positive control well and the three samples with hemoglobin concentrations of 50 g / L, 100 g / L, and 150 g / L. After microplate reading, according to the kit instructions, the judgment value was calculated as 2.1 × the average OD value of the negative wells. The calculated average OD value of the negative wells was 0.339 (0.357, 0.318, and 0.343 for the three wells), and the judgment value was 0.7119. The judgment value was compared with the average OD values ​​of four gradient hemolytic negative samples (20 g / L, 50 g / L, 100 g / L, and 150 g / L). It was found that the average OD values ​​of the three samples with hemoglobin concentrations of 50 g / L, 100 g / L, and 150 g / L were all higher than the judgment value (50 g / L: 0.955, 100 g / L: 1.223, 150 g / L: 2.586). Moreover, the OD value readings increased with increasing hemoglobin concentration, confirming that the impact of hemolysis on ELISA detection increases with the degree of hemolysis. Combined with the kit instructions, which clearly state that hemolysis of the specimen will affect the final test results, it is evident that the ELISA method is not suitable for forensic postmortem blood samples with severe hemolysis.

Claims

1. A method for detecting allergens in drug-induced anaphylactic shock, characterized in that, include: S1. The sample to be tested is contacted with a capture reagent conjugated with anti-IgE antibody to obtain an incubation mixture; S2. Add protease to the incubation mixture obtained in step S1 for enzymatic digestion, and collect the supernatant; S3. Perform LC-MS analysis on the supernatant obtained in step S2, and obtain the allergens in drug-induced anaphylactic shock through the LC-MS results; The samples to be tested are: hemolyzed blood from the corpse and / or pericardial fluid from the corpse.

2. The method as described in claim 1, characterized in that, In step S1, the capturing reagent includes at least one of latex microspheres and magnetic microspheres; Preferably, the surface of the capturing reagent contains carboxyl groups.

3. The method as described in claim 1, characterized in that, Step S1 includes: After contacting the sample to be tested with the capture reagent conjugated with anti-IgE antibody, it was incubated at 37°C for 2-5 hours. After incubation, the supernatant was discarded by the first centrifugation to obtain the incubation mixture. Preferably, the conditions for the first centrifugation are 10000-15000 r for 5-10 min; Preferably, after the supernatant is discarded by the first centrifugation, the process further includes a washing step; Preferably, the cleaning step includes: adding PBS and sonicating, followed by a second centrifugation; Preferably, the conditions for the second centrifugation are 10000-15000 r for 5-10 min; Preferably, the cleaning is repeated 2-8 times.

4. The method as described in claim 1, characterized in that, In step S2, the protease is papain; Preferably, the protease hydrolysis is performed under the following conditions: incubation at 37°C for 1-5 hours. Preferably, after the enzymatic hydrolysis incubation is completed, deionized water is added, and the supernatant is collected after a third centrifugation. Preferably, the conditions for the third centrifugation are 10000-15000 r for 5-10 min.

5. The method as described in claim 1, characterized in that, In step S3, before performing LC-MS detection on the supernatant obtained in step S2, a preprocessing step is also included, which includes: The supernatant was mixed with a methanol-acetonitrile mixture, and the supernatant was retained after a fourth centrifugation. Preferably, in the methanol-acetonitrile mixture, the volume ratio of methanol to acetonitrile is (1.5-2.5):(0.5-1.5). Preferably, the conditions for the fourth centrifugation are 11000-16000 r for 2-8 min.

6. The method as described in claim 1, characterized in that, In step S3, the liquid chromatography conditions are at least one of the following a1-a4: a1. Column type: Any one of the following: C4, C8, C12, and C18 column types; a2. Mobile phase: 15-30 mmol ammonium acetate and 0.05-0.2% formic acid buffer: acetonitrile; a3. Flow rate: 0.1-0.5 ml / min; a4. Injection volume: 2-10 µL.

7. The method as described in claim 1, characterized in that, In step S3, the mass spectrometry conditions are at least one of the following b1-b4: b1. ESI ion source; b2. Drying airflow rate: 5-20 L / min; b3. Atomizer pressure 40-60 psi; b4. Multiple Response Monitoring (MRM) mode.

8. The method as described in claim 1, characterized in that, The drugs include at least one of the following: cefoperazone, sulbactam, levofloxacin, clindamycin, sodium creatine phosphate, ribavirin, ceftriaxone, cimetidine, aztreonam, gentamicin, ambroxol, moxifloxacin, dexamethasone, and pazufloxacin.

9. The application of the method as described in claims 1-8 is at least one of the following c1-c2: c1. Detecting allergens in the sample that bind to specific IgE; c2. Sensitizing drugs in subjects who have experienced anaphylactic shock due to forensic drug testing.

10. A system for detecting allergens in drug-induced anaphylactic shock, characterized in that, include: 1) Inspecting components; 2) Data processing components; 3) Output components; The detection component is configured to: contact the sample to be tested with a capture reagent conjugated with anti-IgE antibody to obtain an incubation mixture, add protease to the incubation mixture, take the supernatant after enzymatic digestion, and perform LC-MS detection on the supernatant; The data processing component is configured to: a. receive detection data of the sample to be tested; b. store the detection data of the sample to be tested; c. compare the sample to be tested and the drug standard with: i. retention time, ii. qualitative ion and iii. ion pair abundance ratio; d. if all three indicators i-iii are consistent with the drug standard, then the sensitizer of the sample to be tested is the drug corresponding to the drug standard. The result output component is configured to output the allergens of the sample to be tested; The samples to be tested are hemolyzed blood from corpses and / or pericardial fluid from corpses.

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