Method for detecting drug antibody in specimen

Abstract

Provided is a method for detecting a drug antibody in a specimen. In the present disclosure, the drug antibody is detected by flow cytometry for the first time. During the detection, self cells of a patient are used as indicator cells. Moreover, nine systems are set up, including: a blank system, a negative control system, a positive control system, a self—control system, a drug—sensitized self—cell control system, a drug—antibody reaction detection system, an enzyme—sensitized self—control system, an enzyme—reaction drug detection system, and an enzyme—reaction drug control system. The method avoids interference from autoantibodies and irregular antibodies of red blood cells, and also avoids a false positive result of detection caused by combination of enzymes or drugs with self red blood cells. The method for detecting a drug antibody can simultaneously detect an affinity of the drug antibody to red blood cells and identify a type of the drug antibody.

Description

TECHNICAL FIELD [OOOl]The present disclosure belongs to the technical field of antibody detection, and in particular relates to a method for detecting a drug antibody in a specimen. BACKGROUND

[0002] The immunogenicity of a drug refers to an ability of the drug to induce an immune response or immunerelated events against itself or related proteins. Immunogenicity has wideranging implications. This mechanism may have no effect, or may affect an efficacy of the drug, and may even cause serious side effects in more serious cases. When an immune response occurs, the drug acts as an antigen and stimulates the body to produce drug antibodies. The drug antibodies can seriously affect the efficacy of the drug, or destroy (kill) blood cells in the human body. In severe cases, the drug antibodies may lead to hemolytic anemia, or result in ineffective blood transfusion and even life threatening.

[0003] For detection methods of the drug antibody, it is generally necessary to pretreat a specimen to be tested using highdensity enrichment magnetic beads. This pretreatment realizes the detection of a total antidrug antibody (ADA) in a form including a drugADA_compleX in the specimen, thereby overcoming the influence of high concentration drug proteins in the specimen and improving the drug resistance of an ADA.detection method. However, the pretreatment has complicated scheme and high cost. During the detection by enzymelinked immunosorbent assay (ELISA), after a general drug antigen is combined with an antibody, colorimetric determination is conducted, and a concentration is calculated by a curve equation. However, the method has poor sensitivity. The detection can also be conducted by a microcolumn gel method. In this method, the drug antigen is combined with the antibody, and obtained results are observed through a gel column. However, the method still has subjective determination differences and uncontrollable factors of sensitivity. Moreover, the method uses self selected allogeneic cells as indicator cells, and cannot avoid interference from autoantibodies and irregular antibodies of red blood cells.

[0004] _Accordingly, there is an urgent need for a drug antibody detection method with simple protocols, accurate results, and shielding from various interferences. SUMMARY

[0005] A_purpose of the present disclosure is to provide a method for detecting a drug antibody in a specimen. The method can avoid interference from alloantibodies (that is, irregular antibodies of red blood cells) and autoantibodies in the specimen, and realize simple, efficient, and accurate detection. Moreover, the method can complete detection of an affinity of the drug antibody to red blood cells and identification of a type of the drug antibody.

[0006] The present disclosure provides a method for detecting' a drug antibody in a specimen, including' the following steps: preparing at least nine reaction systems; conducting flow cytometry on the at least nine reaction systems sequentially; and determining presence or absence of the drug antibody, a type of the drug antibody, and an affinity of the drug antibody to red blood cells according to results of the flow cytometry; where

[0007] the nine reaction systems include a blank system, a negative control system, a positive control system, a self control system, a drugsensitized selfcell control system, a drugantibody reaction detection system, an enzyme sensitized selfcontrol system, an enzymereaction drug detection system, and an enzymereaction drug control system; [OOO8]the blank system includes self cells and phosphate buffered saline (PBS) (1X, pH=7.4, the same below);

[0009] the negative control system. includes RhD negative cells, IgG antiD serum, and the PBS;

[0010] the positive control system includes RhD positive cells, the IgG antiD serum, and the PBS;

[0011] the selfcontrol system includes the self cells, the PBS, and self plasma;

[0012] the drugsensitized selfcell control system includes the self cells, the PBS, and a drug liquid;

[0013] the drugantibody reaction detection system includes drugsensitized self cells, the drug liquid, and the self plasma;

[0014] the enzymesensitized. selfcontrol system. includes enzymesensitized self cells, the PBS, and the self plasma;

[0015] the enzymereaction drug detection system includes the enzymesensitized self cells, the drug liquid, and the self plasma;

[0016] the enzymereaction drug control system includes the enzymesensitized self cells, the drug liquid, and the PBS; and

[0017] the drug liquid is a drug solution that produces a drug antibody to be tested.

[0018] Preferably, the self plasma is derived from a serum specimen or a plasma specimen of a patient with a medication history.

[0019] Preferably, the self plasma is a supernatant obtained after conducting centrifugation on the serum specimen or the plasma specimen of the patient with the medication history; and

[0020] the centrifugation is conducted at 10,000 rpm for 15 min to 20 min.

[0021] Preferably, the self cells are self red blood cells of the patient with the medication history.

[0022] Preferably, a preparation method of the drug sensitized self cells includes: mixing a normal saline instead of partial plasma with self cells of a patient and a drug at a medication concentration of the human body, and conducting incubation to obtain the drugsensitized self cells.

[0023] Preferably, a preparation method of the enzyme sensitized. self cells includes: mixing self cells of a patient with za papain. solution. at an equal volume, and conducting incubation to obtain the enzymesensitized self cells.

[0024] Preferably, the self cells, the drugsensitized self cells, and the enzymesensitized self cells each are configured into a 5% suspension during preparation of respective systems.

[0025] Preferably, each 150 uL of the blank system includes 50 uL of the self cells (5%) and 100 uL of the PBS;

[0026] each 300 uL of the negative control system includes 100 uL of the RhD negative cells (5%), 100 uL of the IgG antiD serum, and 100 uL of the PBS;

[0027] each 300 uL of the positive control system includes 100 uL of the RhD positive cells (5%), 100 uL of the IgG antiD serum, and 100 uL of the PBS;

[0028] each 300 uL of the selfcontrol system includes 100 uL of the self cells (5%), 100 uL of the PBS, and 100 uL of the self plasma;

[0029] each 300 uL of the drugsensitized selfcell control system includes 100 uL of the self cells (5%), 100 uL of the PBS, and 100 uL of the drug liquid;

[0030] each 300 uL of the drugantibody reaction detection system includes 100 uL of the drugsensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the self plasma;

[0031] each. 300 uL of the enzymesensitized. selfcontrol system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the PBS, and 100 uL of the self plasma;

[0032] each 300 (HJ of the enzymereaction drug detection system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the self plasma; and

[0033] each 300 uL of the enzymereaction drug control system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the PBS.

[0034] Preferably, the method further includes the following steps before the flow cytometry is conducted: conducting incubation on each of the reaction systems at 37°C, mixing with a fluorescentlylabeled antibody diluent obtained by conducting dilution on 500 uL of the PBS and 5 uL of a fluorescent stock solution at 1:(50100), and conducting incubation in the dark.

[0035] Preferably, a type of an induction drug of the drug antibody includes a drugsensitized type, a drugadded type, and an enzymetreated drugantibody immune complex type.

[0036] Beneficial effects: in the present disclosure, the drug antibody is detected by flow cytometry for the first time. During the detection, self cells of a patient are used as indicator cells. Moreover, three systems are set up, including a selfcell control, an enzymesensitized control, and a drugsensitized control. The method avoids interference from autoantibodies and irregular antibodies of red blood cells, and also avoids a false positive result of detection caused by combination of enzymes or drugs with self red blood cells.

[0037] The method for detecting a drug antibody can simultaneously detect an affinity of the drug antibody to red blood cells and identify a type of the drug antibody. The affinity refers to an ability of the drug or the drug antibody or a drugdrug antibody complex to bind to red blood cells, and can also be called a strength of adhesion to a surface of the red blood cell. The affinity is reflected by a reading of a fluorescence intensity (namely a Mean value). If the fluorescence is strong, the affinity is strong, and vice versa. If the Mean value of a positive control or a test tube is greater than the Mean value of a negative control, the specimen to be tested is positive (namely successful combination. of antigenantibody). The experiment has controllable process and credible results. .All detection systems are compared. with the Mean of a negative control system. If the Mean value of the detection system.is greater than the Mean value of the negative control, the specimen to be tested is determined as positive. If the mean value of the drugantibody reaction detection system is greater than the mean values of the selfcontrol system and the drugsensitized selfcell control system, and the mean value of the enzymereaction drug detection system is greater than the mean values of the enzymesensitized self control system and the enzymereaction drug control system, the specimen to be tested is determined to be positive for the drug antibody. The method has strong sensitivity and intuitive data readout, thereby improving a detection rate of weak antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To describe the technical solutions in the examples of the present disclosure or in the prior art more clearly, the accompanying drawings required for the examples will be briefly described below.

[0039] FIG. 1 shows a specimen addition pattern of the systems;

[0040] FIGS. 2AI show flow cytometry diagrams of the nine specimen injection systems;

[0041] FIGS. 3AC show flow cytometry results of Tubes 0 to 2 in Example 1;

[0042] FIGS. 4AC show flow cytometry results of Tubes 3 to 5 in Example 1;

[0043] FIGS. 5AC show flow cytometry results of Tubes 6 to 8 in Example 1;

[0044] FIGS. 6AC show flow cytometry results of Tubes 1, 2, and 5 in Example 2;

[0045] FIGS. 7AC show flow cytometry results of Tubes 1, 2, and 5 in Example 3;

[0046] FIGS. 8AC show flow cytometry results of Tubes 1, 2, and 5 in Example 4;

[0047] FIGS. 9AD show flow cytometry results of Tubes 1, 2, 5, and 7 in Example 5; and

[0048] FIGS. 10AC show flow cytometry results of Tubes 1, 2, and 5 in Example 6. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] The present disclosure provides a method for detecting' a drug antibody in a specimen, including' the following steps: preparing at least nine reaction systems; conducting flow cytometry on the at least nine reaction systems sequentially; and determining presence or absence of the drug antibody, a type of the drug antibody, and an affinity of the drug antibody to red blood cells according to results of the flow cytometry; where

[0050] the nine reaction systems include a blank system, a negative control system, a positive control system, a self control system, a drugsensitized selfcell control system, a drugantibody reaction detection system, an enzyme sensitized selfcontrol system, an enzymereaction drug detection system, and an enzymereaction drug control system;

[0051] the blank system includes self cells and PBS;

[0052] the negative control system. includes RhD negative cells, IgG antiD serum, and the PBS;

[0053] the positive control system includes RhD positive cells, the IgG antiD serum, and the PBS;

[0054] the selfcontrol system includes the self cells, the PBS, and self plasma;

[0055] the drugsensitized selfcell control system includes the self cells, the PBS, and a drug liquid;

[0056] the drugantibody reaction detection system includes drugsensitized self cells, the drug liquid, and the self plasma;

[0057] the enzymesensitized. selfcontrol system. includes enzymesensitized self cells, the PBS, and the self plasma;

[0058] the enzymereaction drug detection system includes the enzymesensitized self cells, the drug liquid, and the self plasma;

[0059] the enzymereaction drug control system includes the enzymesensitized self cells, the drug liquid, and the PBS; and

[0060] the drug liquid is a drug solution that produces a drug antibody to be tested.

[0061] In the present disclosure, the self plasma is preferably derived from a serum specimen or a plasma specimen of za patient with a ndication history. In an example, the serum specimen or the plasma specimen of the patient with the medication history is preferably centrifuged at 10,000 rpm for 15 min to 20 min, and an obtained supernatant is collected to obtain the self plasma.

[0062] In the present disclosure, the self cells are preferably patient's self red blood cells. When a system is prepared, the patient's self red blood cells are washed with a normal saline, prepared into a 5% suspension, and the 5% suspension is used to prepare the system. The 5% suspension is preferably using 1XPBS, pH=7.4 as a diluent, and adding 50 uL of packed red blood cells into 1,000 uL of the PBS, to obtain. a suspension of the red blood cells with a concentration of 5%. The washing is preferably conducted 5 times. A. preparation. method. of the drugsensitized. self cells includes preferably: mixing a normal saline instead of partial plasma with self red blood cells of a patient and a drug at a medication concentration of the human body, and conducting incubation to obtain the drugsensitized self cells. Preferably, the normal saline is used to replace 55% (v / v) of the plasma, mixed with 45% (v / v) of the packed red blood cells and the drug, and then incubated at 37°C for 1.5 h.to obtain the 5% suspension, so as to prepare the system. A preparation method of the enzymesensitized self cells includes preferably: mixing self red blood cells of a patient with za papain. solution. at an equal volume, and conducting incubation to obtain the enzymesensitized self cells. The incubation is preferably conducted at 37°C for 15 min, and an obtained product is washed 5 times with a large amount of normal saline to obtain the 5% suspension, so as to prepare the subsequent system.

[0063] In the present disclosure, since each of the systems are used for flow cytometry detection, the systems each are prepared in a centrifuge tube. Therefore, each of the systems may also be numbered, including:

[0064] Tube 0: 50 uL of the self cells (5%) and 100 uL of the PBS;

[0065] Tube 1: the negative control system includes 100 uL of the RhD negative cells (5%), 100 uL of the IgG antiD serum, and 100 uL of the PBS;

[0066] Tube 2: the positive control system includes 100 uL of the RhD positive cells (5%), 100 uL of the IgG antiD serum, and 100 uL of the PBS;

[0067] Tube 3: the selfcontrol system includes 100 uL of the self cells (5%), 100 uL of the PBS, and 100 uL of the self plasma;

[0068] Tube 4: the drugsensitized selfcell control system includes 100 uL of the self cells (5%), 100 uL of the PBS, and 100 uL of the drug liquid;

[0069] Tube 5: the drugantibody reaction detection system includes 100 uL of the drugsensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the self plasma;

[0070] Tube 6: the enzymesensitized selfcontrol system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the PBS, and 100 uL of the self plasma;

[0071] Tube 7: the enzymereaction. drug' detection. system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the self plasma; and

[0072] Tube 8: the enzymereaction drug control system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the PBS.

[0073] In the present disclosure, after Tubes 0 to 8 are prepared to obtain the systems, respectively, the method further includes preferably: after conducting incubation at 37°C, adding a fluorescentlylabeled antibody diluent obtained by conducting dilution on 500 uL of the PBS and 5 uL of a fluorescent stock solution at 1:(50100), mixing, conducting incubation in the dark, followed by determination on a machine. More preferably, after adding specimens into each of the tubes, the specimens each are incubated at 37°C for 1.5 h, washed 3 times with PBS, and added with 100 uL of the fluorescentlylabeled antibody diluent (the fluorescent antibodies are preferably added with FITC labeled goat antihuman IgG Fc, and diluted 50 to 100 times before adding the specimens), incubated in the dark at 37°C for 30 mini5 min, suspended in the PBS, placed in the dark at a room temperature, and determined within 2 h.

[0074] In the present disclosure, a procedure for onboard detection includes preferably: adjusting a voltage with Tube 0 (self cells), acquiring data after gating, and defaulting an optical channel voltage and a photoelectric magnification according to a current state of a flow cytometer 'used; loading a specimen into a test tube, acquiring 50 uL of the specimen, collecting 10,000 red blood cells within a set gate, and collecting a fluorescence intensity of a drug antigenantibody conjugate on a surface of each red blood cell.

[0075] In.the present disclosure, Tube 0 is used for "gating" during the flow cytometry detection. Tube 1 is a negative control tube for an antigenantibody binding reaction. Tube 2 is a positive control tube for the antigenantibody binding reaction. Tube 3 is a "zero" control tube for autoantibodies. Tube 4 is a "zero" control tube for drug sensitized self cells. Tube 5 is a detection tube for drug antibody identification. and. cell affinity. Tube 6 is a "zero" detection tube of the enzymesensitized self cells, as a control. Tube 7 is an enzymedependent immunocomplex detection tube for drug antibody identification and cell affinity. Tube 8 is a "zero" control tube for enzyme and drugsensitized self cells.

[0076] By using the method of the present disclosure, the detection of the affinity of the drug antibody to red blood cells and the identification of the type of the drug antibody can be completed simultaneously. The affinity of the drug antibody to red blood cells is detected.by the flow cytometry, so as to understand a destructive power of the drug antibody to red blood cells, that is, the lethality. This method is suitable for drug antibody identification of a drug sensitized type (such as piperacillin, amoxicillin) and a drugadded type (such as ceftriaxone, vancomycin), as well as antibody detection of EHI enzymetreated drugantibody immune complex type. The method can avoid interference from alloantibodies (that is, irregular antibodies of red blood cells) and autoantibodies in the patient's plasma. The affinity refers to an ability of the drug' or the drug antibody or a drugdrug antibody complex to bind to red blood cells, and can also be called a strength of adhesion to a surface of the red blood cell. The affinity is reflected by a reading of a fluorescence intensity (namely a Mean value). If the fluorescence is strong, the affinity is strong, and vice versa. The drugdrug antibody binds to red blood cells, sensitizes red blood cell membranes or changes a structure of the red blood cell membrane. Some antibodies can also stimulate complement to destroy red blood cells and cause red blood cell lysis, which is the socalled lethality. That is, the stronger binding capacity and more bound drug drug antibody results in easier destruction of the red blood cells.

[0077] For drugsensitized drugs (such as piperacillin and amoxicillin kits): when there are drug antibodies in the patient's plasma, when medication is conducted again, the drug can bind to the drug antibody when it enters the blood. However, the antibody in the plasma does not bind to the red blood cells after being combined with the drug, so it is necessary for the drug to sensitize the red blood cells first (since the drugsensitized red blood cells may also cause fluorescence enhancement, in the present disclosure, a control tube (Tube 4) with drug sensitization and not being bound to antibody is set as a reference base for fluorescence readings). The plasma to be tested is added. If there is a corresponding drug antibody in the plasma, the drug antibody can combine with the red blood cells sensitized to the drug, and the fluorescence is enhanced.

[0078] For drugadded drugs (such as ceftriaxone and vancomycin kits): after the formation of immune complexes, the drugs free in plasma can bind to the surface of red blood cells after binding to antibodies. The enzyme reagents can enhance the ability of the drugantibody complex to bind to red blood cells, and an enzymeinvolved assay is suitable for the detection of immune complexes. Moreover, in the present disclosure, the enzymeinvolved assay is set in each experiment, and is suitable for the detection of patients who take multiple drugs, so as not to miss a certain drug antibody.

[0079] In order to further illustrate the present disclosure, the nethod. for detecting a. drug antibody ill a specimen provided in the present disclosure is described in detail below in conjunction. with. the accompanying' drawings and examples, which are not to be construed as limiting the scope of protection of the present disclosure.

[0080] In the present disclosure, the specimens of each example are prepared through a same operation after collection:

[0081] Self plasma: the patients serum or plasma specimen is centrifuged at 10,000 rpm for 15 min to 20 min, and a supernatant is collected for later use.

[0082] Patient's self cells: the cells are washed 5 times with a normal saline and prepared into a 5% suspension.

[0083] Drugsensitized red blood cells: the patient's self cells are mixed with normal saline instead of plasma (55%) + packed red blood cells (45%) + drug (according to human drug concentration), mixed, incubated at 37°C for 1.5 h, and prepared into a 5% suspension using PBS.

[0084] Enzymesensitized red blood cells: the patient's self cells are washed 5 times with a normal saline, mixed well with an equal volume of packed red blood cells + an equal volume of an enzyme solution, incubated at 37°C for 15 min, washed 5 times with a large amount of the normal saline, and prepared into a 5% suspension with PBS. The sampling rules for each tube are shown in FIG. 1:

[0085] Tube 0: 50 uL of the self cells (5%) and 100 uL of the PBS;

[0086] Tube 1: the negative control system includes 100 uL of the RhD negative cells (5%), 100 uL of the IgG antiD serum, and 100 uL of the PBS;

[0087] Tube 2: the positive control system includes 100 uL of the RhD positive cells (5%), 100 uL of the IgG antiD serum, and 100 uL of the PBS;

[0088] Tube 3: the selfcontrol system includes 100 uL of the self cells (5%), 100 uL of the PBS, and 100 uL of the self plasma;

[0089] Tube 4: the drugsensitized selfcell control system includes 100 uL of the self cells (5%), 100 uL of the PBS, and 100 uL of the drug liquid;

[0090] Tube 5: the drugantibody reaction detection system includes 100 uL of the drugsensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the self plasma;

[0091] Tube 6: the enzymesensitized selfcontrol system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the PBS, and 100 uL of the self plasma;

[0092] Tube 7: the enzymereaction. drug' detection. system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the self plasma; and

[0093] Tube 8: the enzymereaction drug control system includes 100 uL of the enzymesensitized self cells (5%), 100 uL of the drug liquid, and 100 uL of the PBS.

[0094] .After adding specimens according to the rules of different tubes, each obtained system is incubated at 37°C for 1.5 h and washed 3 times with PBS. 100 uL of a diluted fluorescent antibody is added (the fluorescent antibody is preferably FITClabeled goat antihuman IgG Fc according to the purpose of detection, and then diluted 50 to 100 times before adding the specimens), and incubated in the dark at 37°C for 30 mini5 min. An obtained product is suspended in PBS, placed in the dark at a room.temperature, and determined on a machine within 2 h. Tube 0 (self cells) is used to adjust a voltage and conduct gating to collect data, and an optical channel voltage and a photoelectric magnification are defaulted according to a current state of a flow cytometer used. A specimen is loaded into a test tube, 50 uL of the specimen is acquired, 10,000 red blood cells are collected within a set gate, and a fluorescence intensity of a drug antigenantibody conjugate is collected. on a surface of each red blood cell.

[0095] Example 1

[0096] Wu, a patient at the Municipal People's Hospital, was tested for the drug antibody to ceftriaxone. The medication history of ceftriaxone was 4 d (2022.2.2023), and the drug antibody to ceftriaxone was detected (by flow cytometry: Attune WXT).

[0097] The results were shown in FIGS. 2AI to FIGS. 5A-C and Table 1, the drug antibody to ceftriaxone was positive, and was a drugadded type.

[0098] Table 1 Mean value statistics and result determination of each system

[0099] NO . 0 1 2 3 4 6 8 value -------

[0100] Example 2

[0101] Jiao, a patient from the Third Hospital of the City, was tested on the 5th day of piperacillin administration in this medical history (2022.1.1418), and the drug antibody to piperacillin was detected (by flow cytometer: Attune WXT). The results were shown in FIGS. 6AC, the drug antibody to piperacillin was weakly positive.

[0102] Example 3

[0103] Qin, a patient from Children's Hospital, was tested on the 7th day of amoxicillin administration in this medical history (2021.11.1016), and the drug antibody to amoxicillin was detected (by flow cytometer: BD FASCanto II). The results were shown in FIGS. 7AC, the drug antibody to amoxicillin was strongly positive.

[0104] Example 4

[0105] Peng, a patient from the Third Hospital of the City, was tested on the 6th day of piperacillin administration in this medical history (2021.11.1419), and the drug antibody to piperacillin was detected (by flow cytometer: BD FASCanto II). The results were shown in FIGS. 8AC, the drug antibody to piperacillin was positive.

[0106] Example 5

[0107] Wu, a patient from the Third Hospital of the City, was tested on the 7th day of ceftriaxone administration in this medical history (2021.12.28), and the drug antibody to ceftriaxone was detected (by flow cytometer: Attune WXT). The results were shown in FIGS. 9AD, the drug antibody to ceftriaxone was weakly positive by enzyme reaction.

[0108] Example 6

[0109] Gao, a patient in the Eighth Affiliated Hospital, Sun Yatsen University, had no medication history (after the test result was negative, the inventors communicated with the hospital again and learned that the patient began to be treated with piperacillin only after blood was collected and sent for examination in the hospital). The drug antibody to piperacillin was detected (by flow cytometer: Attune WXT). The results were shown in FIGS. 10AC, the drug antibody to piperacillin was negative.

[0110] Although the above example has described the present disclosure in detail, it is only a part of, not all of, the examples of the present disclosure. Other examples may also be obtained by persons based on the example without creative efforts, and all of these examples shall fall within the protection scope of the present disclosure.

Claims

1. Method for detecting a drug antibody in a sample comprising the following steps: preparing at least nine reaction systems to perform flow cytometry in sequence on at least nine reaction systems; and the determination of the presence or absence of the drug antibody body, a type of the drug antibody and a affinity of the drug antibody for red blood cells according to the results of flow cytometry; where bee the nine reaction systems a blank system, a ne positive control system, a positive control system, a self-control system, a medicine sensitized. self-cell control system a. drug the antibody reaction detection system, an enzyme sensitized self-control system, an enzyme reaction drug detection system and an enzyme reaction drug control system include: the blank system own cells and phosphate buffered saline solution (PBS) includes; the negative control system RhD negative cells, IgG antiD serum and the PBS includes; the positive control system RhD-positive cells, the IgGantiD serum and the PBS includes; the own control system, the own cells, the PBS and the own plasma includes; the drug-sensitized self cell control system the own cells, the PBS and a cure medium fluid includes; l7 the drug antibody response detection system drug-sensitized own cells, the drug includes the fluid and its own plasma; the enzyme-sensitized self-control system and enzyme-sensitized own cells, the PBS and the own plasma includes; the enzyme reaction drug detection system the and zyme-sensitized own cells, the drug fluid includes dust and its own plasma; the enzyme reaction drug control system the and zyme-sensitized own cells, the drug fluid substance and the PBS; and the drug liquid is a drug solution that produces a drug antibody to be tested.

2. The method of claim 1 wherein the egg plasma is obtained from a serum sample or urine sample sample from a patient with a medication history.

3. Method according to claim 2, wherein the own plasma is a supernatant obtained after performing centrifugation of the serum sample or plasma sample of the patient with the medication history; and centrifuging for 15 to 20 minutes is performed at 10,000 rpm.

4. The method of claim 1, wherein the own cells own red blood cells are from the patient with the medication history.

5. The method of claim 1, wherein a preparation drug sensitized egg mode of action Gene cells involves: mixing a normal saline solution instead of plasma from a patient with the patient's own cells the patient and a medicine in a medication center tie of the human body, and performing incu bation to destroy the drug-sensitized own cells to acquire.

6. The method of claim 1, wherein a preparation mechanism of action of the enzyme-sensitized own cells involves mixing a patient's own cells with a papain solution in an equal volume, and perform from incubation cm1 the enzyme-sensitized own cells available.

7. A method according to claim 1, 5 or 6, wherein the own cells, the drug-sensitized own cells and the enzyme-sensitized own cells every tide the preparation of the respective systems in a 5% suspension can be configured. The method of claim 7, wherein each 150 µl of the blank system 50 µl of the own cells (5%) and 100 ul of PBS includes; every 300 pl of the negative control system 100 pl of the RhD negative cells (5%), 100 µl of the IgG anti Dserpm and 100 pl of the PBS includes; every 300 pl of the positive control system 100 pl of the RhD positive cells (5%), 100 µl of the IgG anti Includes serum and 100 ul of PBS; every 300 pl of the self-control system 100 pl of the own cells (5%), 100 µl of the PBS and 100 µl of the own plasma includes; every 300 pl of the drug-sensitized own cell control system 100 µl of the own cells (5%), 100 µl of the PBS and 100 µl of the drug solution includes; every 300 pl of the medicine antibody response detection system 100 pl of the drug desensitized own cells (5%), 100 µl of the ge includes drug solution and 100 ul of the patient's own plasma; every 300 pl of the enzyme-sensitized own control system 100 pl of the enzyme-sensitized egg gene cells (5%), 100 µl of the PBS and 100 µl of the egg gene plasma includes; every 300 pl of the enzyme reaction drug detection system 100 pl of the enzyme sensitized own cells (5%), 100 µl of the drug medium fluid and 100 pl of the own plasma; and every 300 pl of the enzyme reaction drug monitoring system 100 pl of the enzyme sensitized own cells (5%), 100 µl of the drug medium fluid and 100 µl of PBS.

9. The method of claim 8, further comprising the following steps before performing flow cytometry performed: performing incubation on each of the reaction systems at 37°C, mixing with a fluorescently labeled antibody diluent, and performing incubation tie in the dark.

10. The method of claim 1, wherein a type of an induction drug of the drug antibody a drug-sensitized type, a drug-added type and an enzyme-treated ge drug antibody immune complex type includes. oooo e1Tube2Tube3Tube4Tube5Tube6Tube7Tube8DtivelsulrumulSulativerole)RhDpositivecells100ulD-serum100ulPBS100ul(Positivecontroltube)Selfcells100ulPBS100ulSelf serum100ul(Self-controltube)Selfcells100ulPBS100ulDrugliquid100ul(Drug-sensitizedself-cellcontroltube)Drug-sensitizedselfcells100ulDrugliquid100ulSerum1 00ul(Drug-antibodyreactiondetectiontube)Enzyme-sensitizedselfcells100ulPBS100ulSerum100ul(Enzyme-sensitizedself-controltube)Enzyme-sensitizedselfcells10 0ulDrugliquid100ulSerum100ul(Enzyme-reactiondrugdetectiontube)Enzyme-sensitizedselfcells100ulDrugliquid100ulPBS100ul(Enzyme-reactiondrugcontroltube)FIG.1 FIG. 2A