Immunomagnetic bead method detection method and kit based on universal PK

Abstract

The invention relates to an immunomagnetic bead method detection method based on general type PK and a kit, and solves the technical problems that in the prior art, an immunomagnetic bead method is adopted for PK detection, the kit is poor in universality, and the repeatability and the stability of a detection result need to be improved. Detection is carried out according to the processes of magnetic bead-sample incubation, two times of washing, detection antibody incubation and signal acquisition. The method is especially suitable for PK index accurate detection scenes in biotechnology drug research and development and clinical application monitoring.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to an immunomagnetic bead detection method and kit based on universal PK. Background Technology

[0002] Pharmacokinetic (PK) studies are a core component of drug development, clinical application monitoring, and personalized treatment guidance. By precisely analyzing the concentration changes of drugs in biological samples, PK studies provide crucial data support for drug dosage optimization, efficacy evaluation, and safety monitoring. With the rapid development of biotechnology drugs, the requirements for sensitivity, specificity, detection efficiency, and result consistency in PK testing are increasingly stringent, and traditional detection techniques have gradually revealed many limitations.

[0003] Currently, commonly used techniques in the field of PK testing include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and conventional immunomagnetic bead assay. Among these, while ELISA is relatively simple to operate, it has a long detection cycle (4-6 hours per test), involves many steps (more than 8 steps including sample loading, incubation, and washing), and is susceptible to human factors (sample loading errors, insufficient washing, etc., leading to batch-to-batch CV ≥ 15%). Although HPLC has good specificity (specificity ≥ 90%), sample pretreatment is complex (requiring more than 5 steps including extraction and purification), requires high professional skills from operators, and has high instrument maintenance costs (annual maintenance cost ≥ 50,000 yuan), making it unsuitable for grassroots laboratories and high-throughput testing scenarios.

[0004] Immunomagnetic bead assays, with their advantages of high specific surface area and strong specific binding ability, have been gradually applied in pharmacokinetic (PK) detection. However, existing immunomagnetic bead detection technologies still have significant shortcomings: on the one hand, the kits have poor versatility, with most designed for specific drug targets (e.g., only suitable for detecting a certain type of monoclonal antibody drug), making it difficult to adapt to the PK detection needs of different types of drugs (monoclonal antibodies, small molecule drugs, fusion proteins, etc.), leading to increased detection costs (cost of a single-target kit ≥ 2000 RMB / kit) and extended development cycles (development cycle of a single kit ≥ 6 months); the intra-assay CV of the detection results is ≥ 8%, and the inter-assay CV is ≥ 12%, requiring improvements in the repeatability and stability of the detection results. For example, Chinese invention patent CN111579772A discloses a detection method and kit based on immunomagnetic beads, but it only optimizes the magnetic bead coating process for specific antibody detection and does not solve the problem of kit versatility. Summary of the Invention

[0005] This application aims to address the technical problems of poor kit universality and the need to improve the repeatability and stability of test results in the existing technology of PK detection using immunomagnetic beads, and provides an immunomagnetic bead detection method and kit based on universal PK.

[0006] This application provides an immunomagnetic bead detection method based on a universal PK, which is performed according to the following process: first, the sample is incubated with magnetic beads, then washed for the first time, then incubated with detection antibodies, then washed for the second time, and finally the signal is detected and the results are analyzed.

[0007] Preferably, it includes the following steps: Step S1, incubation of sample with magnetic beads: Add the sample, detection antibody reagent, and universal immunomagnetic bead reagent to each well of three 96-well plates respectively; Place a 96-well plate containing immunomagnetic bead reagent on the platform, with the 96-well plate located below the magnetic rod sleeve module; When the magnetic rod sleeve module and the magnetic rod module are combined, the first lifting drive mechanism and the second lifting mechanism operate synchronously, so that the magnetic rod module and the magnetic rod sleeve module descend synchronously into the 96-well plate containing immunomagnetic bead reagent, and the magnetic rod sleeve of the magnetic rod sleeve module adsorbs the magnetic beads. In step S2, the first lifting drive mechanism and the second lifting mechanism operate synchronously to raise the magnetic rod module and the magnetic rod sleeve module synchronously, and remove the magnetic rod module and the magnetic rod sleeve module from the 96-well plate containing the immunomagnetic bead reagent. Step S3: Place the 96-well plate containing the sample on the platform and below the magnetic rod sleeve module; the first lifting drive mechanism and the second lifting mechanism operate synchronously to make the magnetic rod module and the magnetic rod sleeve module descend synchronously into the 96-well plate containing the sample. Step S4: The first lifting mechanism moves the magnetic rod module to rise, and the magnetic rod module separates from the magnetic rod sleeve module. Next, a tapping operation is performed to mix the magnetic beads in the sample solution. Then, the mixture is shaken and incubated at room temperature. Step S5, First Wash: The first lifting mechanism lowers the magnetic rod module, assembling it with the magnetic rod sleeve. The magnetic rod sleeve attracts magnetic beads. Next, the first and second lifting mechanisms move synchronously, raising the magnetic rod module and the magnetic rod sleeve module. The magnetic rod sleeve module with attached magnetic beads is then removed from the 96-well plate containing the sample. Next, a 96-well round-bottom plate for cleaning is placed on a platform below the magnetic rod sleeve module. Next, the first and second lifting mechanisms move synchronously, lowering the magnetic rod module and the magnetic rod sleeve module into the 96-well round-bottom plate for cleaning. Next, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module. Finally, the sample is tapped and washed. In step S6, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve, which then attracts the magnetic beads. Next, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve module, and the magnetic rod sleeve module with the magnetic beads is removed from the 96-hole round bottom plate for cleaning. Step S7, Detect antibody incubation: A 96-well plate containing the detection antibody reagent is placed on the platform, below the magnetic rod sleeve module. Next, the first and second lifting mechanisms operate synchronously, causing the magnetic rod module and magnetic rod sleeve module to descend into the 96-well plate containing the detection antibody reagent, with the magnetic rod sleeve containing magnetic beads inserted into the plate. Next, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module. Next, the plate is tapped to mix the magnetic beads in the detection antibody solution. Finally, the plate is shaken and incubated at room temperature. Step S8, second wash: The first lifting mechanism lowers the magnetic rod module, assembling it with the magnetic rod sleeve, which then attracts magnetic beads. Next, the first and second lifting mechanisms move synchronously, raising the magnetic rod module and the magnetic rod sleeve simultaneously. Next, a 96-hole round-bottom plate for cleaning is placed on the platform, below the magnetic rod sleeve module. Next, the first and second lifting mechanisms move synchronously, lowering the magnetic rod module and the magnetic rod sleeve into the 96-hole round-bottom plate. Next, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve. Finally, the device is tapped and washed. In step S9, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve, which then attracts the magnetic beads. Next, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve module, and the magnetic rod sleeve module with the magnetic beads is then removed from the 96-hole round bottom plate for cleaning. Step S10, Signal Detection and Result Analysis: After adding substrate reagent to each well of the ELISA plate, the ELISA plate is placed on the platform. Next, the first lifting drive mechanism and the second lifting mechanism operate synchronously, causing the magnetic rod module and the magnetic rod sleeve module to descend into the ELISA plate simultaneously. Next, the first lifting mechanism operates to raise the magnetic rod module, separating the magnetic rod module from the magnetic rod sleeve module. Next, the plate is tapped, and the magnetic beads are in the substrate reagent, causing the substrate reagent to develop color. After the color development is completed, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve. Then, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve synchronously. Finally, place the ELISA plate into the ELISA reader and read the value on the reader.

[0008] Preferably, the 96-well plate is a 96-well PCR plate.

[0009] This application also provides a kit comprising a universal PK detection immunomagnetic bead reagent, a detection antibody reagent, a washing buffer, a substrate reagent, a sample diluent, and a standard; the universal PK detection immunomagnetic bead reagent has a universal linker group modified on the surface of the magnetic beads, a coating solution containing a sodium tritiate catalyst, and a blocking solution containing mannouronic acid.

[0010] Preferably, the surface of the magnetic beads is modified with carboxyl groups; the blocking solution contains 0.9% w / v NaCl, 0.1% w / v mannuluronic acid, and 1% w / v BSA phosphate.

[0011] The beneficial effects of this invention are that it improves versatility; the universal magnetic beads are suitable for detecting more than three types of drugs, expanding the applicable range by 2-5 times compared to existing technologies. It can also be adapted to detect multiple types of drugs by changing the targeting antibody.

[0012] Achieve high throughput, improve efficiency, and meet the rapid detection needs of large-scale samples. Reduce detection costs. Improve detection stability.

[0013] The repeatability and stability of the test results are improved, significantly reducing intra-batch and inter-batch CV values. When testing 500 clinical samples, the consistency of the test results is ≥98%; for 10 consecutive batches, the intra-batch CV is ≤3.5%, and the inter-batch CV is ≤7%.

[0014] The linear correlation coefficient of the standard curve is ≥0.995, which meets the requirements for high-precision detection.

[0015] It is especially suitable for the accurate detection of PK indicators in biotechnology drug development and clinical application monitoring.

[0016] Further features and aspects of this disclosure will be clearly described in the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of a magnetic rod module and a magnetic rod sleeve combined together, with the magnetic rod sleeve module inserted into a PCR plate. Figure 2 This is a schematic diagram of a 96-hole round-bottom perforated plate; Figure 3 This is a schematic diagram showing the magnetic rod sleeve module and the magnetic rod module connected to the first lifting mechanism and the second lifting mechanism, respectively. Figure 4 This is a schematic diagram showing the magnetic rod sleeve module and the magnetic rod module combined together, with the magnetic rod sleeve placed on the magnetic rod. Figure 5 This is a schematic diagram of the second lifting mechanism connected to the reciprocating motion drive mechanism; Figure 6 These are the test results; Figure 7 These are results from traditional manual testing; Figure 8 It is a standard curve; Figure 9 This is a schematic diagram of the structure and binding principle of universal immunomagnetic beads; Figure 10 This is a flowchart of the detection process.

[0018] Explanation of symbols in the diagram: 1. Magnetic rod and magnetic rod sleeve assembly, 2. Magnetic rod sleeve, 3. PCR plate, 4. 96-well round bottom plate, 5. First lifting mechanism, 6. Second lifting mechanism, 7. Magnetic rod module, 8. Magnetic rod sleeve module, 9. Reciprocating motion drive mechanism. Detailed Implementation

[0019] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] The specific embodiments described below are merely preferred embodiments of this application, and the scope of protection of this application is not limited thereto. Those skilled in the art can make modifications or variations based on the principles, concepts, and spirit of this application, and the resulting technical solutions should all be covered within the scope of protection of this application.

[0021] like Figure 3 As shown, the magnetic rod module 7 is connected to the first lifting mechanism 5, which can raise or lower the magnetic rod module 7. The power source of the first lifting mechanism 5 can be a motor. The magnetic rod sleeve module 8 is connected to the second lifting mechanism 6, which can raise or lower the magnetic rod sleeve module 8. The power source of the second lifting mechanism 6 can also be a motor. The magnetic rod module 7 includes 96 magnetic rods, and the magnetic rod sleeve module 8 includes 96 magnetic rod sleeves. The number of magnetic rod sleeves corresponds to the number of magnetic rods.

[0022] like Figure 4 As shown, the first lifting mechanism 5 moves the magnetic rod module 7 downwards, and the magnetic rod of the magnetic rod module 7 is inserted into the magnetic rod sleeve of the magnetic rod sleeve module 8. The magnetic rod module 7 and the magnetic rod sleeve module 8 are combined together to form an assembly. Figure 1 As shown, the magnetic rod module 7 and the magnetic rod sleeve module 8 are combined together, and the magnetic rod sleeve is inserted into the well of the PCR plate 3.

[0023] The general-purpose immunomagnetic bead reagents involved in the examples can be KanryBead. TMA universal immunomagnetic bead reagent, with magnetic beads of 1-3 μm size (manufacturer: Kangrui Bio, model: RMB001), surface-modified with carboxyl groups, achieving a coating efficiency of 92%. The coating solution uses 0.1 mol / L potassium phosphate buffer (pH 7.4) with 0.02% sodium tritiate added as a catalyst. After coating, a phosphate blocking solution containing 0.9% w / v NaCl, 0.1% w / v mannuluronic acid, and 1% w / v BSA is used, and the mixture is blocked at 37°C for 1.5 h. This reagent offers multi-target compatibility and high coating efficiency.

[0024] KanryAb can be used as a detection antibody reagent. TM Detection antibody reagent: Mouse anti-human IgG Fcκ monoclonal antibody-HRP (Manufacturer: KanryBio, Model: RAB002), concentration: 1 mg / mL; stored in PBS (pH 7.2-7.6) at -25 to -15°C.

[0025] KanryLab substrate reagents can be used. TM Substrate reagent: TMB ultrasensitive colorimetric solution (manufacturer: KanryBio, model: RCM007), store at 2-8℃, signal decay of 8% after 24 hours at room temperature, store at 2-8℃.

[0026] The washing buffer can be a phosphate buffer containing 0.05% Tween-20 (manufacturer: KanryBio, model: RCM007), stored at room temperature.

[0027] The following detection methods and kits are compatible with drug types such as monoclonal antibodies, small molecules, and fusion proteins.

[0028] refer to Figure 10 The immunomagnetic bead detection method based on universal PK includes the following steps: Step S1, incubation of sample with magnetic beads: Add 100 μL of sample, 100 μL of detection antibody reagent, and 100 μL of universal immunomagnetic bead reagent to each well of three 96-well PCR plates respectively. Place the 96-well PCR plate containing the immunomagnetic bead reagent on the platform. The 96-well PCR plate is located below the magnetic rod sleeve module. With the magnetic rod sleeve module and the magnetic rod module together (the magnetic rod sleeve is on the magnetic rod), the first lifting drive mechanism and the second lifting mechanism operate synchronously, so that the magnetic rod module and the magnetic rod sleeve module descend synchronously into the 96-well PCR plate containing the immunomagnetic bead reagent. The magnetic rod sleeve is inserted into the well of the 96-well PCR plate, and the magnetic rod sleeve of the magnetic rod sleeve module adsorbs the magnetic beads.

[0029] In step S2, the first lifting drive mechanism and the second lifting mechanism operate synchronously, causing the magnetic rod module and the magnetic rod sleeve module to rise simultaneously, thus removing the magnetic rod module and the magnetic rod sleeve module from the 96-well PCR plate containing immunomagnetic bead reagent. The magnetic rod sleeve module is attached with magnetic beads. 96 samples are extracted at a time.

[0030] Step S3: Place the 96-well PCR plate containing the sample on the platform, below the magnetic rod sleeve module; the first lifting drive mechanism and the second lifting mechanism operate synchronously, so that the magnetic rod module and the magnetic rod sleeve module descend synchronously into the 96-well PCR plate containing the sample.

[0031] In step S4, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module (the magnetic rod detaches from the magnetic rod sleeve). The magnetic rod sleeve remains in the well of the 96-well PCR plate. Next, a tapping operation is performed, causing the magnetic rod sleeve to reciprocate and mix the magnetic beads in the sample solution. Then, the plate is shaken (300 rpm) and incubated at room temperature for 60 minutes to form a sample-antibody immune complex.

[0032] The specific mechanism for implementing the tapping operation can adopt the tapping structure in invention application CN119776114A, or the tapping structure in invention patent CN111273032B.

[0033] Step S5, First Wash: The first lifting mechanism lowers the magnetic rod module, assembling it with the magnetic rod sleeve (the magnetic rod is inserted into the sleeve). The sleeve attracts magnetic beads. Next, the first and second lifting mechanisms move synchronously, raising the magnetic rod module and sleeve simultaneously. The sleeve with attached magnetic beads is then removed from the 96-well PCR plate containing the sample. Next, a 96-well round-bottom plate for cleaning is placed on the platform, below the magnetic rod sleeve module. Next, the first and second lifting mechanisms move synchronously, lowering the magnetic rod module and sleeve into the 96-well round-bottom plate for cleaning, with the sleeve containing magnetic beads inserted. Next, the first lifting mechanism raises the magnetic rod module, separating it from the sleeve, while the sleeve remains in the 96-well round-bottom plate. Finally, the plate is tapped three times and washed three times with 200 μL of washing buffer per well, each wash lasting 60 seconds. The instrument monitors the liquid level in real time to ensure thorough washing and complete extraction of waste liquid.

[0034] In step S6, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve (the magnetic rod is inserted into the magnetic rod sleeve), and the magnetic rod sleeve attracts the magnetic beads. Next, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve module synchronously, and the magnetic rod sleeve module with the magnetic beads is removed from the cleaning perforated plate.

[0035] Step S7, Detect antibody incubation: A 96-well PCR plate containing detection antibody reagents is placed on the platform, below the magnetic rod sleeve module. Next, the first and second lifting mechanisms operate synchronously, causing the magnetic rod module and magnetic rod sleeve module to descend into the 96-well PCR plate containing the detection antibody reagents, with the magnetic rod sleeve containing magnetic beads inserted into the plate. Next, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module, while the magnetic rod sleeve remains in the 96-well PCR plate containing the detection antibody reagents. Next, a tapping operation is performed to mix the magnetic beads in the detection antibody solution. Finally, the plate is shaken at 400 rpm and incubated at room temperature for 60 minutes to form an antibody-sample-antibody "sandwich" complex.

[0036] Step S8, second wash: The first lifting mechanism lowers the magnetic rod module, assembling it with the magnetic rod sleeve (the magnetic rod is inserted into the sleeve). The sleeve attracts magnetic beads. Next, the first and second lifting mechanisms move synchronously, raising the magnetic rod module and sleeve simultaneously. The sleeve with attached magnetic beads is then removed from the 96-well PCR plate containing the detection antibody reagent. Next, a 96-well round-bottom plate for cleaning is placed on the platform, below the magnetic rod sleeve module. Next, the first and second lifting mechanisms move synchronously, lowering the magnetic rod module and sleeve into the 96-well round-bottom plate for cleaning, with the sleeve containing magnetic beads inserted. Next, the first lifting mechanism raises the magnetic rod module, separating it from the sleeve, while the sleeve remains in the 96-well round-bottom plate. Finally, the plate is tapped three times and washed three times with 200 μL of washing buffer per well, each wash lasting 60 seconds.

[0037] In step S9, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve (the magnetic rod is inserted into the magnetic rod sleeve), and the magnetic rod sleeve attracts the magnetic beads. Next, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve module synchronously, and the magnetic rod sleeve module with the magnetic beads is removed from the 96-hole round bottom plate for cleaning.

[0038] Step S10, Signal Detection and Result Analysis: After adding 100 μL of substrate reagent to each well of the ELISA plate, the plate is placed on the platform. Next, the first and second lifting mechanisms operate synchronously, causing the magnetic rod module and magnetic rod sleeve module to descend into the ELISA plate, with the magnetic rod sleeve containing the magnetic beads inserted into the wells. Next, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module, while the magnetic rod sleeve remains in the well. Then, a tapping operation is performed, dissolving the magnetic beads in the substrate reagent, which then develops color. After color development, the first lifting mechanism lowers the magnetic rod module, reassembling it with the magnetic rod sleeve module (magnetic rod inserted into the magnetic rod sleeve), with the magnetic rod sleeve adsorbing the magnetic beads. Finally, the first and second lifting mechanisms operate synchronously, raising the magnetic rod module and magnetic rod sleeve module simultaneously, removing the magnetic rod sleeve module containing the magnetic beads from the ELISA plate. Finally, place the ELISA plate into the ELISA reader and read and analyze the absorbance (ELISA labeling detects absorbance at 450nm–630nm, and fluorescence labeling detects excitation light signal); calculate the sample concentration based on the standard curve.

[0039] Samples used for fitting the standard curve, quality control samples, and test samples: Humanized anti-HER-2 monoclonal antibody (manufacturer: KanryBio, concentration: 3 mg / mL), stored at 2~8℃; Sample dilution buffer: Casein buffer (manufacturer: KanryBio, model: RBC002), stored at 2~8℃. The standard curve is shown below. Figure 8 As shown, R² = 0.999.

[0040] KanryAb TM Capture antibody reagent for coupling magnetic beads: Mouse anti-human IgG Fc monoclonal antibody (MouseAnti-Human IgG Fc mAb) (manufacturer: KanryBio, model: RAB001), concentration: 1 mg / mL; stored in PBS (pH 7.2-7.6) at 2-8°C.

[0041] Test results as follows Figure 6 As shown, compared to Figure 7 The results of the traditional manual test shown indicate a significant improvement in detection sensitivity, which is 0.1 ng / mL; the CV value is reduced, with an intra-batch CV of 3.2% and an inter-batch CV of 6.8%.

[0042] The detection throughput has been significantly increased, improving efficiency and meeting the needs for rapid detection of large-scale samples. It also reduces detection costs and improves detection stability.

[0043] After the kit was stored at 4°C for 12 months, the detection sensitivity remained at 0.1 ng / mL, and the signal attenuation was ≤10%.

[0044] For mechanisms that implement the tapping operation, the following can be used: Figure 5 The reciprocating motion drive mechanism 9 is shown. The second lifting mechanism 6 is connected to the reciprocating motion drive mechanism 9. The operation of the reciprocating motion drive mechanism 9 causes the second lifting mechanism 6 to reciprocate, which in turn causes the magnetic rod sleeve to reciprocate.

Claims

1. A detection method based on universal PK using immunomagnetic beads, characterized in that, The process is as follows: first, the sample is incubated with magnetic beads; second, it is washed for the first time; third, the detection antibody is incubated; fourth, it is washed for the second time; and finally, the signal is detected and the results are analyzed.

2. The immunomagnetic bead detection method based on universal PK according to claim 1, characterized in that, Includes the following steps: Step S1, incubation of sample with magnetic beads: Add the sample, detection antibody reagent, and universal immunomagnetic bead reagent to each well of three 96-well plates respectively; Place a 96-well plate containing immunomagnetic bead reagent on the platform, with the 96-well plate located below the magnetic rod sleeve module; When the magnetic rod sleeve module and the magnetic rod module are combined, the first lifting drive mechanism and the second lifting mechanism operate synchronously, so that the magnetic rod module and the magnetic rod sleeve module descend synchronously into the 96-well plate containing immunomagnetic bead reagent, and the magnetic rod sleeve of the magnetic rod sleeve module adsorbs the magnetic beads. In step S2, the first lifting drive mechanism and the second lifting mechanism operate synchronously to raise the magnetic rod module and the magnetic rod sleeve module synchronously, and remove the magnetic rod module and the magnetic rod sleeve module from the 96-well plate containing the immunomagnetic bead reagent. Step S3: Place the 96-well plate containing the sample on the platform and below the magnetic rod sleeve module; the first lifting drive mechanism and the second lifting mechanism operate synchronously to make the magnetic rod module and the magnetic rod sleeve module descend synchronously into the 96-well plate containing the sample. Step S4: The first lifting mechanism moves the magnetic rod module to rise, and the magnetic rod module separates from the magnetic rod sleeve module. Next, a tapping operation is performed to mix the magnetic beads in the sample solution. Then, the mixture is shaken and incubated at room temperature. Step S5, First Wash: The first lifting mechanism lowers the magnetic rod module, assembling it with the magnetic rod sleeve. The magnetic rod sleeve attracts magnetic beads. Next, the first and second lifting mechanisms move synchronously, raising the magnetic rod module and the magnetic rod sleeve module. The magnetic rod sleeve module with attached magnetic beads is then removed from the 96-well plate containing the sample. Next, a 96-well round-bottom plate for cleaning is placed on a platform below the magnetic rod sleeve module. Next, the first and second lifting mechanisms move synchronously, lowering the magnetic rod module and the magnetic rod sleeve module into the 96-well round-bottom plate for cleaning. Finally, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module. Next, pat and wash; In step S6, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve, which then attracts the magnetic beads. Next, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve module, and the magnetic rod sleeve module with the magnetic beads is removed from the 96-hole round bottom plate for cleaning. Step S7, Detect antibody incubation: A 96-well plate containing detection antibody reagents is placed on a platform, below the magnetic rod sleeve module. Next, the first lifting drive mechanism and the second lifting mechanism operate synchronously, causing the magnetic rod module and the magnetic rod sleeve module to descend synchronously into the 96-well plate containing the detection antibody reagents, and the magnetic rod sleeve with attached magnetic beads is inserted into the 96-well plate containing the detection antibody reagents. Next, the first lifting mechanism operates to raise the magnetic rod module, separating the magnetic rod module from the magnetic rod sleeve module. Next, tap to mix the magnetic beads in the detection antibody solution; then shake and incubate at room temperature. Step S8, second wash: The first lifting mechanism lowers the magnetic rod module, assembling it with the magnetic rod sleeve, which then attracts magnetic beads. Next, the first and second lifting mechanisms move synchronously, raising the magnetic rod module and the magnetic rod sleeve simultaneously. Then, a 96-hole round-bottom plate for cleaning is placed on the platform, below the magnetic rod sleeve module. Next, the first and second lifting mechanisms move synchronously, lowering the magnetic rod module and the magnetic rod sleeve into the 96-hole round-bottom plate. Finally, the first lifting mechanism raises the magnetic rod module, separating it from the magnetic rod sleeve module. Next, pat and wash; In step S9, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve, which then attracts the magnetic beads. Next, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve module, and the magnetic rod sleeve module with the magnetic beads is then removed from the 96-hole round bottom plate for cleaning. Step S10, Signal Detection and Result Analysis: After adding substrate reagent to each well of the ELISA plate, the ELISA plate is placed on the platform; next, the first lifting drive mechanism and the second lifting mechanism operate synchronously to lower the magnetic rod module and the magnetic rod sleeve module into the ELISA plate simultaneously; next, the first lifting mechanism operates to raise the magnetic rod module and separate the magnetic rod module from the magnetic rod sleeve module. Next, tap the magnetic beads in the substrate reagent, and the substrate reagent will develop color. After the color development is completed, the first lifting mechanism lowers the magnetic rod module and combines it with the magnetic rod sleeve. Then, the first lifting drive mechanism and the second lifting mechanism move synchronously to raise the magnetic rod module and the magnetic rod sleeve synchronously. Finally, place the ELISA plate into the ELISA reader and read the value on the reader.

3. The immunomagnetic bead detection method based on universal PK according to claim 2, characterized in that, The 96-well plate is a 96-well PCR plate.

4. A reagent kit, characterized in that, This includes universal PK detection immunomagnetic bead reagents, detection antibody reagents, washing buffer, substrate reagents, sample diluents, and standards; The universal PK detection immunomagnetic bead reagent has a universal linker group modified on the surface of the magnetic beads, a coating solution containing sodium tritiate catalyst, and a blocking solution containing mannuronic acid.

5. The reagent kit according to claim 3, characterized in that, The surface of the magnetic beads is modified with carboxyl groups; the sealing solution contains 0.9% w / v NaCl, 0.1% w / v mannuluronic acid, and 1% w / v BSA phosphate.

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