Blood type specificity IgG antibody typing detection reagent card, kit and preparation method thereof

Abstract

The invention relates to a blood type specificity IgG antibody typing detection reagent card, a kit and a preparation method thereof. The blood type specificity IgG antibody typing detection reagent card comprises a plurality of accommodating holes and information identifiers, magnetic beads coated with anti-erythrocyte antibodies, acridinium ester-labeled anti-human IgG antibodies of different subtypes, and a working solution are respectively arranged in the accommodating holes. The preparation method comprises the following steps: preparing magnetic beads coated with anti-erythrocyte antibodies and different subtypes of anti-human IgG antibodies marked by acridinium ester; respectively mixing the magnetic beads coated with the anti-erythrocyte antibodies and the acridinium ester-labeled anti-human IgG antibodies of different subtypes with a protection solution; and the information identification is arranged on the outer side of the containing hole. The blood type specificity IgG antibody typing detection kit comprises the reagent card, a pre-excitation solution and an excitation solution. The reagent card is high in specificity, easy and convenient to operate and suitable for large-scale clinical detection, and the detection efficiency and accuracy are improved.

Description

Technical Field

[0001] The present application relates to the technical field of blood type detection, and in particular to a blood type specific IgG antibody typing detection reagent card, a kit and a preparation method thereof. Background Art

[0002] Hemolytic disease of the newborn (HDN) is a self-limiting immune syndrome common in fetuses and newborns. Its main feature is hemolytic phenomenon. It is usually caused by blood type incompatibility between mother and fetus, especially in the ABO blood type system, Rh blood type system and MN blood type system.

[0003] Among the many factors that lead to HDN, the IgG antibody subtype in the serum of pregnant women with high titer IgG antibodies is closely related to the occurrence and severity of hemolytic disease of the newborn. Especially when IgG1 or IgG3 subtype antibodies are present in the serum of pregnant women, the incidence of hemolytic disease of the newborn is higher and the condition may be more serious. Therefore, timely detection and treatment are particularly important.

[0004] In the process of detecting blood type-specific antibodies, the microcolumn gel method is currently mainly relied on. This method uses a specific ratio of dextran gel and is divided into two layers, "reaction pool" and "separation pool" in a special microcolumn. Under the action of centrifugal force, free red blood cells that are not bound to antibodies can pass through the gel layer, while red blood cells bound to or agglutinated with specific antibodies cannot pass through. Through this separation method, the reaction state of red blood cells with different antibodies can be effectively judged.

[0005] However, it is worth noting that although the microcolumn gel method performs well in the detection of blood type-specific antibodies, it can currently only detect total IgG levels and cannot type specific IgG. This means that although we know that IgG antibodies exist in the serum of pregnant women, we cannot know exactly which subtype of IgG antibodies is at work. This to some extent limits our in-depth understanding of the pathogenesis and treatment of neonatal hemolytic disease. Therefore, further development and improvement of IgG subtype detection technology is of great significance to improving the diagnosis and treatment of HDN. Summary of the invention

[0006] In order to solve the problem that specific IgG antibodies cannot be accurately typed when blood type-specific antibody detection is performed using the microcolumn gel method in the related art, the present application provides a blood type-specific IgG antibody typing detection reagent card, a kit and a preparation method thereof, aiming to achieve accurate detection and typing of blood type-specific IgG antibody subtypes in the serum of pregnant women, thereby more accurately predicting the occurrence and severity of neonatal hemolytic disease, and providing strong support for clinical diagnosis and treatment.

[0007] In the first aspect, the present application provides a blood type specific IgG antibody typing detection reagent card using the following technical solution: A blood type specific IgG antibody typing detection reagent card comprises a plurality of containing holes and information labels; magnetic beads coated with anti-erythrocyte antibodies, acridinium ester-labeled anti-human IgG antibodies of different subtypes, and working fluid are respectively arranged in the containing holes.

[0008] By adopting the above technical solution: the reagent card is reasonably designed, the operation steps are simple and quick, and it is suitable for large-scale clinical application. It relies on specific antibody-coated magnetic beads and acridinium ester labeling technology to achieve accurate detection and typing of specific IgG antibody subtypes, which can more accurately predict the occurrence and severity of neonatal hemolytic disease, and provide strong support for clinical diagnosis and treatment.

[0009] Preferably, the anti-human IgG antibodies of different subtypes include at least three of anti-human IgG1 subtype antibodies, anti-human IgG2 subtype antibodies, anti-human IgG3 subtype antibodies, and anti-human IgG4 subtype antibodies.

[0010] By adopting the above technical scheme: each subtype antibody has unique antigen binding characteristics and functions, so the use of specific antibodies for typing detection can greatly reduce the possibility of non-specific binding and cross-reaction, improve the accuracy of the test results, and detect these subtypes at the same time to fully understand the distribution of IgG antibodies in pregnant women's serum, providing richer information for clinical use; Being able to obtain accurate IgG antibody typing test results can provide strong support for clinicians, guiding them to develop more personalized and effective treatment plans and reduce the incidence and mortality of neonatal hemolytic disease.

[0011] In the second aspect, the present application provides a method for preparing a blood type-specific IgG antibody typing detection reagent card using the following technical solution: A method for preparing a blood type-specific IgG antibody typing detection reagent card, comprising: preparing magnetic beads coated with anti-erythrocyte antibodies and anti-human IgG antibodies of different subtypes labeled with acridinium ester; Magnetic beads coated with anti-erythrocyte antibodies and anti-human IgG antibodies of different subtypes labeled with acridinium esters are respectively mixed with protective solutions to obtain magnetic bead working solutions coated with anti-erythrocyte antibodies and anti-human IgG antibodies of different subtypes labeled with acridinium esters; the above working solutions are respectively placed in containing wells, and information labels are set on the outside to obtain detection cards.

[0012] By adopting the above technical scheme: the prepared blood type-specific IgG antibody typing test reagent card has the advantages of simple operation, accurate results, good stability, etc. It can provide fast and reliable IgG antibody typing test results for clinical practice, help to more accurately predict the occurrence and severity of neonatal hemolytic disease, and provide strong support for clinical diagnosis and treatment.

[0013] Preferably, the preparation method of the magnetic beads coated with anti-erythrocyte antibodies is as follows: The magnetic beads are divided and vortexed to remove the supernatant, and after washing, anti-erythrocyte antibody is added and dispersed in the first buffer, mixed and incubated; After the incubation is completed, the supernatant is removed, the magnetic beads are washed, and redispersed in a second buffer solution. After blocking and incubation, the magnetic beads coated with the anti-erythrocyte antibody are obtained.

[0014] By adopting the above technical scheme: magnetic beads coated with anti-erythrocyte antibodies can be obtained. These magnetic beads have high specificity and stability and are suitable for subsequent IgG antibody typing detection. The method is simple to operate and easy to control, and the prepared magnetic beads can meet the high standards of clinical testing.

[0015] By adopting the above technical solution: optimizing the antibody ratio, the detection reagent's ability to recognize the target IgG subtype can be improved, while reducing nonspecific binding, thereby improving the sensitivity and specificity of the detection.

[0016] Preferably, the preparation method of the acridinium ester-labeled anti-human IgG antibodies of different subtypes is as follows: The anti-human IgG antibodies of different subtypes to be labeled and acridinium ester are mixed and dispersed in a third buffer solution, wrapped with tin foil, and allowed to react in a constant temperature and humidity chamber at 35-37°C for 0.5-1h to obtain a mixture of anti-human IgG antibodies of different subtypes labeled with acridinium ester; then the obtained mixture of anti-human IgG antibodies of different subtypes labeled with acridinium ester is placed in a 50KD ultrafiltration tube for ultrafiltration to remove unreacted acridinium ester, thereby obtaining anti-human IgG antibodies of different subtypes labeled with acridinium ester.

[0017] By adopting the above technical solution: it is possible to efficiently and controllably prepare acridinium ester-labeled anti-human IgG antibodies of different subtypes with high sensitivity and strong specificity, which are suitable for clinical applications such as blood type-specific IgG antibody typing detection.

[0018] Preferably, the anti-human IgG antibodies of different subtypes are anti-human IgG1 antibodies, anti-human IgG2 antibodies, anti-human IgG3 antibodies, and anti-human IgG4 antibodies in a mass ratio of 1:1-1.3:1-1.1:0.8-1.

[0019] In a third aspect, the present application provides a blood type-specific IgG antibody typing detection kit using the following technical solution: A blood type specific IgG antibody typing detection kit comprises the above reagent card and pre-stimulation liquid and stimulation liquid.

[0020] By adopting the above technical solution: the design of the test kit fully considers the user experience, the information identification on the reagent card is clear and concise, and the addition of pre-excitation liquid and excitation liquid is also automated or semi-automated, further improving the detection efficiency.

[0021] Preferably, the method of using the kit is as follows: When performing a sample test, take the reagent card out of the test kit, put it into the reagent card drawer in the fully automatic chemiluminescence immunoassay analyzer, and close the drawer. The information can be automatically read through the information mark on the reagent card; Open the cover of the sample to be tested and place it on the sample rack, and then put the sample rack into the machine; On the test application interface, select "Sample" as the test type, enter the sample information, and select the test item to apply for the test; Click Start to start the test. The instrument automatically completes the aspiration and mixing of the sample and working solution, and incubates. After the incubation is completed, the magnetic bead working solution coated with anti-erythrocyte antibodies is aspirated and placed in the reaction well, and incubated again. After the incubation is completed, the immune complex is washed. After washing, the working solution and acridinium ester-labeled anti-human IgG antibody working solution of different subtypes are added, and incubated at 37°C. After the incubation is completed, the immune complex is washed. After washing, the pre-excitation solution is added to the immune complex and incubated for 1 minute, and then the excitation solution is added and the luminescent signal value is detected. After the test is completed, the sample test results can be viewed.

[0022] By adopting the above technical solution: used in conjunction with a fully automatic chemiluminescence immunoassay analyzer, the entire testing process is highly automated, reducing the errors and contamination risks caused by manual operation.

[0023] In summary, the present application includes at least one of the following beneficial technical effects: 1. The reagent card of this application adopts specific antibody-coated magnetic beads and acridinium ester labeling technology, which can realize accurate typing detection of different subtypes of IgG antibodies (such as IgG1, IgG2, IgG3, IgG4) in the serum to be tested. This specific detection greatly reduces the possibility of non-specific binding and cross-reaction, and improves the accuracy and reliability of the test results; and the accurate typing test results help clinicians to have a more comprehensive understanding of the distribution of IgG antibodies in the serum of pregnant women, and provide a more accurate basis for predicting the occurrence and severity of neonatal hemolytic disease; 2. The reagent card of this application is reasonably designed, the operation steps are simple and fast, and it is suitable for large-scale clinical application. Through the fully automatic chemiluminescence immunoassay analyzer, the steps of sample processing, incubation, cleaning and detection can be automatically completed, which greatly improves the detection efficiency. The use of the pre-excitation solution and the excitation solution in the kit is also automated or semi-automated, which further simplifies the operation process, reduces human errors, and improves the accuracy and stability of the detection; 3. The reagent card and kit of this application provide clinical with rapid and reliable IgG antibody typing test results, which helps clinicians develop more personalized and effective treatment plans. Through accurate typing tests, doctors can more accurately assess the risk of hemolytic disease of the newborn, take timely intervention measures, and reduce the incidence and mortality of the disease. At the same time, comprehensive IgG antibody typing information also provides valuable data support for clinical research, which helps promote the development and innovation of related fields. DETAILED DESCRIPTION

[0024] The present application is further described in detail below in conjunction with the examples. Except for the special instructions below, the raw materials used in the present application are all common commercially available materials. raw material Brand and manufacturer Purpose of Use Specification Anti-human IgG1 subtype antibody Absea Coagulant 2.2mg / mL Anti-human IgG2 subtype antibody Absea Coagulant 2.4mg / mL Anti-human IgG3 subtype antibody Absea Coagulant 2.1mg / mL Anti-human IgG4 subtype antibody Absea Coagulant 2.5mg / mL Magnetic beads For biological Indicator 5％ Acridinium ester Mekate Indicator 2％ Example

[0025] The working solution in the reagent card of this application includes working solution I and working solution II; The magnetic beads coated with anti-erythrocyte antibody working solution and the anti-human IgG antibody working solution of different subtypes labeled with acridinium ester are respectively prepared by mixing the magnetic beads coated with anti-erythrocyte antibody with protective solution I and the anti-human IgG antibody of different subtypes labeled with acridinium ester is mixed with protective solution II.

[0026] The coating buffer was prepared as follows: 1) Use a 500mL graduated cylinder to measure 400mL of ultrapure water, pour it into a 500mL beaker, and stir it on a magnetic stirrer; 2) Weigh 1 g of boric acid and dissolve it completely in a beaker; 3) Calibrate the pH meter and adjust the pH of the solution in the beaker to 8.0 with 1M NaOH solution; 4) Pour the solution in the beaker into a 500mL measuring cylinder and dilute to 500mL; pour into a bottle and store in a refrigerator at 2-8℃.

[0027] The protective solution I was prepared by the following method: 1) Use a 1000mL graduated cylinder to measure 800mL of ultrapure water, pour it into a 1000mL beaker, and stir it on a magnetic stirrer; 2) Weigh 3g Tris and 8g NaCl and completely dissolve them in a beaker; 3) Calibrate the pH meter and adjust the pH of the solution in the beaker to 8.0 (25.1°C) with 1M HCl solution; 4) Weigh 3 g BSA and dissolve it completely in a beaker; 5) Use a pipette to measure 1 mL of Proclin-300 and add it to the solution and mix well; Pour the solution in the beaker into a 1000mL measuring cylinder, dilute to 1000mL, pour into a bottle, and store in a refrigerator at 2-8℃.

[0028] Protective solution II is prepared by the following method: Accurately weigh 0.024 g of potassium dihydrogen phosphate, 0.014 g of disodium hydrogen phosphate, 0.8 g of sodium chloride, and 0.02 g of potassium chloride and put them into a 100 ml beaker. Use a 100 ml measuring cylinder to measure 50 ml of ultrapure water and add it to the beaker to dissolve the solids. Transfer the solution into a 50 ml centrifuge tube and store it in a refrigerator at 2-8 °C.

[0029] Working solution I was prepared by the following method: Accurately weigh 3 g of Tris, 0.8 g of sodium chloride, and 1 g of BSA, and put them into a 100 ml beaker. Use a 100 ml graduated cylinder to measure 50 ml of ultrapure water and add it to the beaker to dissolve the solid. Transfer the solution into a 50 ml centrifuge tube and store it in a refrigerator at 2-8 °C.

[0030] Working solution II was prepared by the following method: Accurately weigh 3g Tris, 0.8g sodium chloride, and 1g casein, and put them into a 100ml beaker. Use a 100ml measuring cylinder to measure 50mL of ultrapure water and add it to the beaker to dissolve the solids. Transfer the solution into a 50ml centrifuge tube and store it in a refrigerator at 2-8℃.

[0031] The magnetic bead working solution coated with anti-human IgG antibodies of different subtypes was prepared by the following method: 1. Magnetic bead washing: (1) Mix the magnetic beads until they are not sticky to the wall. Take four 2 mL centrifuge tubes and pipette 10 mg of magnetic beads into each tube. Put the others back into the 4 °C refrigerator. (2) Vortex the magnetic beads for 30 seconds, place the centrifuge tube on a magnetic stand to separate the magnetic beads, and remove the supernatant; (3) Add 1 ml of coating buffer, vortex for 30 seconds, clean on an ultrasonic cleaner at 7% ultrasonic power for 1 minute, place on a magnetic stand, wait for 1 minute, and then remove the supernatant of the magnetic beads; (4) Vortex and wash the beads twice with the coating buffer prepared above, and remove the supernatant.

[0032] 2. Magnetic bead coating (1) Add coating buffer to the above centrifuge tube, add 0.2 mg of anti-erythrocyte antibody, vortex mix, and then add catalyst and vortex mix; The preparation method of the catalyst is as follows: use a 500mL measuring cylinder to measure 120mL of ultrapure water, pour it into a 500mL beaker, and place it on a magnetic stirrer for stirring; weigh 60g of ammonium sulfate and completely dissolve it in the beaker; calibrate the pH meter and adjust the pH of the solution in the beaker to 9.5 with a 1M NaOH solution; pour the solution in the beaker into a 500mL measuring cylinder, make up to 200mL, pour it into a bottle, and store it in a refrigerator at 2-8°C.

[0033] (2) Incubate the mixture on a rotary mixer at 37°C (20 rpm) for 24 h.

[0034] 3. Magnetic bead blocking (1) Take out the centrifuge tube, adsorb the magnetic beads, and remove the supernatant; (2) Wash the magnetic beads three times with protective solution I, 1 mL each time; (3) Add 1 mL of protective solution I to the centrifuge tube and incubate at 37°C with rotation (20 rpm).

[0035] 4. Magnetic Bead Cleaning and Storage (1) Take out the magnetic beads, adsorb the magnetic beads, and remove the supernatant; (2) Wash three times with protective solution I and remove the supernatant.

[0036] (3) Add protective solution I to the test tube and seal it for storage.

[0037] Acridinium ester labeled anti-human IgG antibody working solutions of different subtypes were prepared by the following method: 1. Take materials: Take 80ul of protective solution II, 2ul of acridinium ester, and anti-human IgG antibodies of different subtypes and add them into a centrifuge tube, wrap it with tin foil, and place it in a constant temperature and humidity chamber at 37°C for reaction for 1 hour.

[0038] 2. Ultrafiltration to remove free acridinium esters: Use a 50KD ultrafiltration tube to ultrafilter to remove unreacted acridinium ester, add 0.2 mL of protective solution II, centrifuge at 8000 rpm at 4°C for 5 min, remove the ultrafiltered solution; repeat the above steps twice again, and transfer the labeled antibody to a new centrifuge tube.

[0039] A blood type-specific IgG antibody typing test reagent card, on which R1 hole, R2 hole, R3 hole, R4 hole and a QR code capable of carrying reagent card related information (reagent name, batch number, expiration date) are provided; The R1 well contains 50ul of magnetic bead working solution coated with anti-erythrocyte antibodies, with a specification of 0.4mg / ml; the R2 well contains 130ul of acridinium ester-labeled anti-human IgG antibody working solution of different subtypes, with a specification of 100ng / ml; the R3 well contains 200ul of working solution I; and the R4 well contains 200ul of working solution II.

[0040] A blood type-specific IgG antibody typing detection kit, which is composed of the above-mentioned reagent card and pre-excitation liquid and excitation liquid, and its specific use method is as follows: When performing sample testing, take the reagent card out of the test kit, put it into the reagent card drawer in the fully automatic chemiluminescence immunoassay analyzer and close the drawer. The reagent-related information (reagent name, batch number, expiration date) can be automatically read through the QR code on the reagent card; Open the cover of the sample to be tested and place it on the sample rack, and then put the sample rack into the machine; On the test application interface, select "Sample" as the test type, enter the sample information, and select the test item to apply for the test; Click Start to start the test. The reagent dosage for each test is 50μl for R1, 100μl for R2, 100μl for R3, and 200μl for R4. Add 10μl of 3% red blood cells to the reaction well. The instrument draws 15ul of the sample to be tested and adds it to R4 well for pre-dilution. Then draw 150ul of the solution from R4 well to R1 well, pipette and mix, and then transfer to the reaction well. Incubate for 24 minutes and wash three times. After washing, add the solution in well R2 and well R3, and incubate at 37°C for 12 minutes. After incubation, wash the immune complex. After washing, add the pre-excitation solution and incubate the immune complex for 1 minute, then add the excitation solution and detect the luminescent signal value. The time from sampling to the result is about 50 minutes.

[0041] Performance testing (I) Physical properties Appearance inspection: Visually inspect the label of the test kit.

[0042] The test results are as follows: product labels should be clearly legible, outer packaging should not be easily worn, and text expressions should be standardized.

[0043] 1. Clinical sample testing The blood type-specific IgG antibody typing detection kit in the embodiment is used to detect the standard sample or the serum (plasma) to be tested, and the detection steps are as follows: (1) After pre-diluting the serum (plasma), the serum (plasma) is mixed with magnetic beads coated with anti-erythrocyte antibodies and then transferred to a reaction well. The red blood cells in the reaction well are captured by the anti-erythrocyte antibodies coated on the magnetic beads. If the serum (plasma) contains IgG antibodies that can bind to red blood cells, a magnetic bead-red blood cell-human IgG antibody complex is formed. The IgG antibodies not bound to the red blood cells are washed away, and then anti-human IgG antibodies of different subtypes labeled with acridinium ester are added. After mixing and incubation, the anti-human IgG antibodies of different subtypes labeled with acridinium ester bind to the IgG antibodies on the red blood cells. (2) After the reaction is completed, the magnetic field attracts the magnetic beads, and the cleaning solution washes away the unbound substances. Then, the pre-excitation solution and the excitation solution are added to the reaction mixture to cause a chemiluminescent reaction.

[0044] (3) The number of photons generated in the reaction is measured by a photomultiplier tube. The number of photons generated is proportional to the amount of IgG in the sample that can cause hemolysis. The amount of IgG in the sample is determined by a built-in calibration curve.

[0045] Result determination: The detection limit of this reagent is: 0.1Ru / mL. If the sample concentration value is <0.1Ru / mL, it is judged as negative; if the sample concentration value is ≥0.1Ru / mL, it is judged as positive.

[0046] 1. Reference substance L0: physiological saline; Reference substance L1: AB type serum (plasma) from a healthy normal person; Reference substances L2, L3, L4: AB type serum (plasma) from a healthy normal person with added anti-D (IgG); Reference substance L5: AB type serum (plasma) from a healthy normal person with added anti-D (IgM).

[0047] 1.1 Blank limit The reference level L0 was used as the sample to be tested and tested 20 times.

[0048] The test results are as follows: All 10 results were negative (<0.05RU / mL).

[0049] 1.2 Detection limit The reference product L2 was used as the sample to be tested and tested 10 times.

[0050] The test results are as follows: All 10 results were positive (≥0.1RU / mL).

[0051] 1.3 Accuracy The reference product L3 was used as the sample to be tested and tested 10 times.

[0052] The test results are as follows: all 10 results were positive, and the relative deviation of the concentration values ​​did not exceed ±30%.

[0053] The reference product L4 was used as the sample to be tested and tested 10 times.

[0054] The test results are as follows: all 10 results were positive, and the relative deviation of the concentration values ​​did not exceed ±30%.

[0055] 2. Specificity detection The reference product L1 was used as the sample to be tested and tested 10 times; The reference product L2 was used as the sample to be tested and tested 10 times; The reference product L5 was used as the sample to be tested and tested 10 times. The test results are listed in the following table: Table: Specificity test results 3. Repeatability test 3.1 Intra-batch reproducibility The reference product L1 was used as the sample to be tested and tested 10 times; The test results are as follows: 10 results were all negative (<0.1RU / mL) and the coefficient of variation of the concentration value CV ≤ 20.0%; The reference product L3 was used as the sample to be tested and tested 10 times; The test results are as follows: all 10 results were positive (≥0.1RU / mL) and the coefficient of variation of the concentration value CV≤20.0%.

[0056] 3.2 Inter-batch repeatability The intra-batch repeatability was tested using three batches of reagent cards; The test results are consistent with the above results.

[0057] 4. Stability test The product was incubated at 37°C for 7 days before undergoing a stability test to measure its physical properties, blank limit, detection limit, accuracy, specificity and batch-to-batch repeatability.

[0058] The test results are consistent with the above results.

[0059] From the above performance test results, the blood type-specific IgG antibody typing test kit in this application shows good performance, specifically including the following aspects: Physical properties and identification: The product label is clearly legible, the outer packaging is not easily worn, and the text is standardized, which ensures the stability and reliability of the test kit during storage, transportation and use.

[0060] Clinical sample testing capabilities: Blank limit: The results of the test with physiological saline (reference substance L0) showed no false positives, verifying the low background interference and sensitivity of the detection system.

[0061] Detection limit: The test results of reference product L2 (healthy normal human AB type serum with anti-D (IgG) added) were all positive, and the signal intensity was higher than the detection limit (0.1Ru / mL), indicating that the kit can accurately detect low concentrations of specific IgG antibodies.

[0062] Accuracy: The test results of reference products L3 and L4 showed that the kit can accurately measure the IgG antibody concentration in the sample, and the relative deviation of the concentration value is within an acceptable range (±30%), which proves the accuracy and reliability of the test results.

[0063] Specificity: By comparing the test results of healthy normal human AB type serum (reference L1), serum with added anti-D (IgG) (reference L2) and serum with added anti-D (IgM) (reference L5), it can be seen that the kit can specifically recognize IgG antibodies, but has no reaction to IgM antibodies, showing good specificity.

[0064] Repeatability: Intra-batch repeatability: Multiple test results of reference products L1 and L3 showed that the negative and positive results were highly stable, and the coefficient of variation of the concentration values ​​was low (CV≤20.0%), indicating that the test kit had good repeatability within the same batch.

[0065] Batch-to-batch repeatability: The test results of different batches of reagent cards are consistent with the above results, which proves the stability and consistency of the kit between different production batches.

[0066] Stability: After the product was incubated at 37°C for 7 days, the performance indicators (physical properties, blank limit, detection limit, accuracy, specificity and batch repeatability) did not change significantly, indicating that the kit has good thermal stability and long-term storage capacity.

[0067] In summary, the blood type-specific IgG antibody typing detection kit in the present application performs well in terms of physical properties, clinical sample detection capability, specificity, repeatability and stability, and can meet the clinical needs for blood type-specific IgG antibody typing detection, providing strong support for clinical diagnosis and treatment.

[0068] The above are all modifications without creative contribution or solutions that obviously constitute technical inspiration that can be made to the present embodiment as needed by technical personnel in this field after reading this specification, but they should be protected by patent law as long as they are within the scope of the claims of this application.

Claims

1. A blood type specific IgG antibody typing test reagent card, used in conjunction with a fully automatic chemiluminescence immunoassay analyzer. It is characterized in that The reagent card includes a plurality of accommodating holes and information labels; The containing holes are respectively provided with magnetic beads coated with anti-erythrocyte antibodies, anti-human IgG antibodies of different subtypes labeled with acridinium ester, and working solution.

2. The blood type-specific IgG antibody typing detection reagent card according to claim 1, It is characterized in that The anti-human IgG antibodies of different subtypes include at least three of anti-human IgG1 subtype antibodies, anti-human IgG2 subtype antibodies, anti-human IgG3 subtype antibodies, and anti-human IgG4 subtype antibodies.

3. A method for preparing a blood type-specific IgG antibody typing detection reagent card according to claim 1 or 2, It is characterized in that include: preparing magnetic beads coated with anti-erythrocyte antibodies and anti-human IgG antibodies of different subtypes labeled with acridinium ester; The magnetic beads coated with anti-erythrocyte antibody and the anti-human IgG antibodies of different subtypes labeled with acridinium ester are respectively mixed with the protective solution to obtain the working solution of the magnetic beads coated with anti-erythrocyte antibody and the working solution of the anti-human IgG antibodies of different subtypes labeled with acridinium ester; The above working fluids are placed in the containing holes respectively, and information marks are set on the outside to obtain a detection card.

4. The method for preparing the blood type-specific IgG antibody typing detection reagent card according to claim 3, It is characterized in that The preparation method of the magnetic beads coated with anti-erythrocyte antibodies is as follows: The magnetic beads are divided and vortexed to remove the supernatant, and after washing, anti-erythrocyte antibody is added and dispersed in the first buffer, mixed and incubated; After the incubation is completed, the supernatant is removed, the magnetic beads are washed, and redispersed in a second buffer solution. After blocking and incubation, the magnetic beads coated with the anti-erythrocyte antibody are obtained.

5. The method for preparing the blood type-specific IgG antibody typing detection reagent card according to claim 3, It is characterized in that The preparation method of the anti-human IgG antibodies of different subtypes labeled with acridinium ester is as follows: The anti-human IgG subtype antibody to be labeled and acridinium ester are mixed and dispersed in a third buffer solution, wrapped with tin foil, and allowed to react in a constant temperature and humidity chamber at 35-37° C. for 0.5-1 h to obtain an anti-human IgG subtype antibody mixture labeled with acridinium ester; Subsequently, the obtained acridinium ester-labeled anti-human IgG subtype antibody mixture is placed in a 50KD ultrafiltration tube for ultrafiltration to remove unreacted acridinium ester, thereby obtaining the acridinium ester-labeled anti-human IgG subtype antibody.

6. The method for preparing the blood type-specific IgG antibody typing detection reagent card according to claim 4, It is characterized in that The anti-human IgG antibodies of different subtypes are anti-human IgG1 antibodies, anti-human IgG2 antibodies, anti-human IgG3 antibodies, and anti-human IgG4 antibodies in a mass ratio of 1:1-1.3:1-1.1:0.8-1.

7. A blood type specific IgG antibody typing detection kit, It is characterized in that It comprises the reagent card as claimed in claim 1 or 2, a pre-stimulation liquid and a stimulation liquid.

8. The blood type-specific IgG antibody typing detection kit according to claim 7, It is characterized in that When used with a fully automatic chemiluminescence immunoassay analyzer, the operation method is as follows: When performing a sample test, take the reagent card out of the test kit, put it into the reagent card drawer in the fully automatic chemiluminescence immunoassay analyzer, and close the drawer. The reagent-related information can be automatically read through the information label on the reagent card; Place the sample to be tested on the sample rack after opening the cover, and then put the sample rack into the machine; On the test application interface, select "Sample" as the test type, enter the sample information, and select the test item to apply for the test; Click Start to begin the test. The instrument automatically completes the aspiration and mixing of the sample and working solution, and incubates. After the incubation is completed, the magnetic bead working solution coated with anti-erythrocyte antibodies is aspirated and placed in the reaction well, and incubated again. After the incubation is completed, the immune complex is washed. After washing, the working solution and acridinium ester-labeled IgG subtype antibody working solution are added, and incubated at 37°C. After the incubation is completed, the immune complex is washed. After washing, the pre-excitation solution is added to incubate with the immune complex for 1 minute, and then the excitation solution is added to detect the luminescent signal value. After the test is completed, the sample test results can be viewed.

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