TAT complex determination method, blocking antibody, detection antibody and kit

Abstract

The invention discloses a TAT complex determination method, a blocking antibody, a detection antibody and a kit, before and after the blocking antibody blocks a 274-site residue of a prothrombin nitrogen terminal, the detection antibody is used as a capture antibody of a TAT complex and acts on a thrombin end close to a thrombin N terminal, so that the TAT complex is detected. After the blocking antibody aiming at the 274-site residue at the nitrogen end of the prothrombin is combined with the prothrombin antibody, the blocking antibody can effectively block the combination of the detection antibody at the N end of the thrombin with the prothrombin, but the blocking antibody aiming at the 274-site residue at the nitrogen end of the prothrombin cannot be combined with the thrombin; therefore, the combination of the detection antibody at the N end of the thrombin to the thrombin is not influenced, and the TAT compound detection process is not interfered by high-concentration prothrombin in the plasma.

Description

Technical Field

[0001] The present application relates to the technical field of TAT complex determination, and specifically to a TAT complex determination method, a blocking antibody, a detection antibody and a kit. Background Art

[0002] After thrombin is formed in the body, its half-life in the blood is only a few seconds, making it difficult to measure directly. The thrombin part quickly combines with antithrombin to form a thrombin-antithrombin III complex. This indicator is a molecular marker that reflects the generation of thrombin. It can sensitively reflect the degree of activation of the coagulation system and directly reflect the activation of the coagulation system. Elevated TAT can predict the risk of thrombosis and recurrence, and the risk of DIC at an early stage.

[0003] Thrombin is Na + A coagulation system protease stimulated by ions, it is a key enzyme involved in the coagulation cascade reaction. After the organism is injured, thrombin quickly forms thrombin from inactive prothrombin through a series of activations. The precursor of thrombin is prothrombin, which is composed of 582 amino residues. After it is activated under certain conditions, the specific parts between the amino acids are disconnected, and the 274 residues at the nitrogen end are cut off. The remaining part can be divided into two peptide chains, called A chain and B chain. Thrombin is a protease composed of 308 amino acids and connected by covalent disulfide bonds. The molecular weight is 37kD. The A chain contains 36 residues, located on the back of the molecule, and plays a stabilizing role in the complete functionality of thrombin, which is called the light chain; the B chain contains 259 residues, has the activity and functional domain of the enzyme, and is called the heavy chain. In other words, the amino acid sequence of prothrombin basically includes the amino acid sequence of thrombin, which will cause antibodies targeting thrombin to bind to thrombin to a large extent, making the development of detection kits difficult.

[0004] There are relatively few TAT detection methods developed so far, mainly the tube chemiluminescence method. As a chemiluminescence immunoassay technology with obvious advantages in immunoassay technology, it has significant advantages in terms of accuracy and sensitivity of the measurement results. At present, the imported TAT complex detection kits that have been put on the market are all suitable for chemiluminescence, but the country still needs related chemiluminescence detection kit products with more stable performance and higher accuracy.

[0005] The reasons why TAT detection kits are difficult to develop are mainly as follows: First, due to the limitations of traditional immunization methods, antibodies against the TAT complex are difficult to develop, so it is difficult to find antibodies with high affinity that recognize the complex; Second, since the proportion of TAT complex in plasma is extremely low, it exists in plasma at a concentration of ng / mL, and plasma contains a large number of monomers of thrombin, prothrombin, and antithrombin III; since the amino acid sequence of prothrombin completely includes the amino acid sequence of thrombin, if no antibody specifically targeting the complex is found, and it is difficult to develop an antibody specifically targeting the spatial structure, it is easy to be blocked by these high-concentration monomers during the detection process, resulting in detection failure or extremely poor detection stability. This is also the main reason why there are no high-performance detection kits in China.

[0006] Since thrombin is derived from prothrombin through enzymatic cleavage, the amino acid sequence of prothrombin includes all the amino acid sequences of thrombin. Most of the antibodies we develop have a large proportion of recognition for the primary amino acid sequence, so the developed antibodies will have a very high proportion of recognizing both thrombin and prothrombin. This will result in a large amount of prothrombin blocking the thrombin-related region of the thrombin-antithrombin III complex when we detect it, which will eventually cause major problems with the stability of the test kit. Summary of the invention

[0007] The purpose of the present application is to provide a TAT complex assay method, blocking antibody, detection antibody and kit, which can effectively block prothrombin during thrombin assay, thereby achieving the purpose of improving detection accuracy.

[0008] To achieve the above objectives, the present application provides the following technical solutions: TAT complex determination method, using blocking antibodies to block prothrombin before and after amino acid 274 of prothrombin, and using detection antibodies acting on the N-terminus of thrombin to detect thrombin, so that the detection antibodies cannot bind to prothrombin.

[0009] Since thrombin is derived from prothrombin through enzymatic cleavage, the amino acid sequence of prothrombin includes all the amino acid sequences of thrombin. Most of the detection antibodies we have developed have a relatively large proportion of recognition for the primary amino acid sequence, so the developed detection antibodies will have a very high proportion of simultaneously recognizing thrombin and prothrombin. This will result in a large amount of prothrombin blocking the thrombin-related region of the thrombin-antithrombin III complex when we detect it. The blocking antibody blocks prothrombin before and after the 274th amino acid of prothrombin, so that the detection antibody will not recognize prothrombin, thereby effectively blocking prothrombin and allowing the detection antibody to recognize thrombin, thereby achieving the purpose of improving detection accuracy.

[0010] A TAT complex assay blocking antibody blocks prothrombin before and after the 274th amino acid of prothrombin, so that the detection antibody will not recognize prothrombin, thereby effectively blocking prothrombin and allowing the detection antibody to recognize thrombin, thereby achieving the purpose of improving detection accuracy.

[0011] A TAT complex detection antibody, the detection antibody acts on the N-terminus of thrombin.

[0012] A TAT complex detection kit comprises a detection antibody acting on the N-terminus of thrombin.

[0013] A method for preparing a TAT complex assay kit, for the above-mentioned TAT complex assay kit, placing the prepared TAT complex assay reagent in the assay kit to obtain the TAT complex assay kit, the preparation of the TAT complex assay reagent comprising: Step 1: Prepare magnetic bead coating buffer; Step 2: Prepare magnetic bead cleaning solution; Step 3: Prepare magnetic bead blocking solution; Step 4: Prepare magnetic bead storage solution; Step 5: prepare magnetic microspheres for first washing, activation, second washing, coating, third washing, blocking, and storage after fourth washing, using magnetic bead washing solution for washing, magnetic bead coating buffer for activation, and magnetic bead blocking solution for blocking. When storing, store the magnetic microspheres in magnetic bead storage solution; Step 6: Use the detection antibody to carry out acridinium ester labeling, and then terminate, purify, elute, and finally store to obtain the TAT complex determination reagent.

[0014] Preferably, the raw materials of the coating buffer include MES, the raw materials of the magnetic bead washing solution include Tris, sodium chloride and Tween-20, the raw materials of the magnetic bead blocking solution include Tris, sodium chloride and BSA, and the raw materials of the magnetic bead preservation solution include Hepes, sodium chloride, BSA, sucrose, Tween-20 and Proclin300.

[0015] Preferably, during activation, a magnetic bead coating buffer is added to the washed magnetic microspheres, and after sufficient mixing, an NHS solution dissolved in pure water is added, and the microspheres are resuspended using a vortex shaker. After sufficient mixing, an EDC solution dissolved in pure water is added, and the microspheres are resuspended using a vortex shaker. After sufficient mixing, the microspheres are shaken and incubated.

[0016] Preferably, during coating, the detection antibody solution and the magnetic bead coating buffer are added to the washed magnetic microspheres, resuspended by a vortex shaking instrument, quickly mixed and then shaken and incubated.

[0017] Preferably, during blocking, BSA diluted with pure water is added to the washed magnetic microspheres, and the microspheres are resuspended using a vortex shaker, fully mixed, and then shaken and incubated.

[0018] Preferably, the acridinium ester label comprises: Step 1: Prepare a labeling buffer, the raw materials of which include disodium hydrogen phosphate and sodium chloride; Step 2: Prepare a stop solution, the raw materials for preparing the stop solution include lysine; Step 3: prepare acridinium ester preservation solution, the raw materials of which include MES, sodium chloride, BSA, sucrose, glycerol, Tween-20 and Proclin300; Step 4: labeling with acridinium ester using detection antibody and labeling buffer, and then performing termination, purification, elution, and finally storage to obtain TAT complex assay reagent. When terminating, add termination solution, and when eluting, add acridinium ester storage solution.

[0019] Compared with the prior art, the beneficial effects of this application are: 1. The TAT complex determination method of the present application, when used, the blocking antibody blocks the vicinity of the 274th residue of the nitrogen end of prothrombin, and the detection antibody serves as a capture antibody of the TAT complex, specifically targeting the action of thrombin near the N-terminus of thrombin. When the blocking antibody targeting the vicinity of the 274th residue of the nitrogen end of prothrombin binds to the prothrombin antibody, the blocking antibody will effectively block the detection antibody at the N-terminus of thrombin from binding to prothrombin. However, since the blocking antibody targeting the vicinity of the 274th residue of the nitrogen end of prothrombin cannot bind to thrombin, it will not affect the binding of the detection antibody at the N-terminus of thrombin to thrombin, so that the TAT complex detection process will not be interfered by the high concentration of prothrombin in the plasma; 2. The blocking antibody for the TAT complex assay of the present application can effectively prevent the detection antibody coated on the chemiluminescent magnetic sphere from binding to free prothrombin in plasma, so that the TAT complex detection process will not be interfered by the high concentration of prothrombin in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the blocking antibody action site for the TAT complex determination of the present application; Figure 2 A comparison chart of the detection results of a TAT complex assay kit proposed in this application and an existing kit; Figure 3 This is a clinical comparison chart between a TAT complex assay kit proposed in this application and existing kits. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application. Embodiment 1

[0022] like Figure 1 As shown, a TAT complex determination method uses a blocking antibody to block prothrombin before and after the 274th amino acid of prothrombin, and uses a detection antibody acting on the N-terminus of thrombin to detect thrombin, so that the detection antibody cannot bind to prothrombin, thereby preventing the TAT complex detection process from being interfered by the high concentration of prothrombin in the plasma. Embodiment 2

[0023] A blocking antibody used in the TAT complex assay method of Example 1, wherein the blocking antibody blocks prothrombin before and after amino acid position 274 of prothrombin. Embodiment 3

[0024] A detection antibody used in the TAT complex determination method of Example 1, wherein the detection antibody acts on the N-terminus of thrombin. Embodiment 4

[0025] A TAT complex assay kit comprises the detection antibody acting on the N-terminus of thrombin of Example 3.

[0026] The preparation method of the TAT complex assay kit of this embodiment is as follows: First, prepare the magnetic beads for coating. Take coating 10mg of magnetic beads as an example. The first step is to prepare 1000ml of coating buffer. The specific formula and conditions are as follows:

[0027] The second step is to prepare 1000ml of magnetic bead cleaning solution. The specific formula and conditions are as shown in the following table.

[0028] The third step is to prepare 1000ml of magnetic bead blocking solution. The specific formula and conditions are as shown in the following table.

[0029] Step 4: Prepare 1000ml of magnetic bead preservation solution. The specific formula and conditions are as shown in the following table.

[0030] After preparation, the first step of cleaning is performed. The specific steps are as follows: take 100 μL of magnetic microspheres and place them in a clean centrifuge tube; add 1000 μL of coating buffer, resuspend with a vortex shaker, mix thoroughly, place on a magnetic stand, and use a pipette to absorb the clear liquid after the magnetic beads are fully attracted by the magnet, and repeat 3 times; Activation after washing: Add 800 μL of coating buffer to the washed magnetic microspheres and mix quickly and thoroughly; then add 100 μL of 10 mg / mL NHS solution (dissolved in pure water), resuspend with a vortex shaker, and mix quickly and thoroughly; finally, add 100 μL of 10 mg / mL EDC solution (dissolved in pure water), resuspend with a vortex shaker, and mix quickly and thoroughly; incubate at 25°C with shaking for 30 minutes; After activation, the second step of washing is performed: the incubated magnetic balls are placed on a magnetic rack, and the clear liquid is sucked out with a pipette after the magnetic beads are fully attracted to the magnet; 1000 μL of coating buffer is added, and the mixture is resuspended with a vortex shaker, and after being fully mixed, the mixture is placed on a magnetic rack, and the clear liquid is sucked out with a pipette after the magnetic beads are fully attracted to the magnet, and this is repeated 3 times; Step 2: Coating after washing: Add X μL of detection antibody A solution to the washed magnetic microspheres, where X is calculated based on the antibody concentration, a total of 0.2 mg, and (1000-X) μL of coating buffer, resuspend with a vortex shaker, quickly and thoroughly mix, and incubate at 25°C for 3 h; After coating, the third step of washing is performed: the incubated magnetic balls are placed on a magnetic rack, and the clear liquid is sucked out with a pipette after the magnetic beads are fully attracted to the magnet; 1000 μL of magnetic bead washing solution is added, and the beads are resuspended with a vortex oscillator, and after being fully mixed, the beads are placed on a magnetic rack, and the clear liquid is sucked out with a pipette after the magnetic beads are fully attracted to the magnet, and this is repeated 3 times; The third step is blocking after washing: add 1000 μL 0.2% BSA (diluted with pure water) to the washed magnetic microspheres, resuspend with a vortex shaker, quickly and thoroughly mix, and incubate at 25°C for 3 h; After blocking, the fourth step of washing is performed: the incubated magnetic balls are placed on a magnetic rack, and the clear liquid is sucked out with a pipette after the magnetic beads are fully attracted to the magnet; 1000 μL of magnetic bead washing solution is added, and the beads are resuspended with a vortex oscillator, and after being fully mixed, the beads are placed on a magnetic rack, and the clear liquid is sucked out with a pipette after the magnetic beads are fully attracted to the magnet, and this is repeated 3 times; Final preservation: Add 1000 μL of magnetic bead preservation solution to the washed magnetic microspheres, resuspend using a vortex shaker, mix quickly and thoroughly, and store in a refrigerator at 2-8°C.

[0031] Next, we will perform acridinium ester labeling, taking 0.2 mg of antibody as an example. First, prepare 1000 ml of labeling buffer. The specific formula and conditions are as shown in the following table.

[0032] Prepare 1000ml of stop solution. The specific formula and conditions are as shown in the following table.

[0033] Prepare 1000 ml of acridinium ester preservation solution. The specific formula and conditions are as shown in the following table.

[0034] After preparation, labeling was performed: X μL of detection antibody A (X is calculated based on the antibody concentration, a total of 0.2 mg) was placed in a clean centrifuge tube, 4.54 μL of 2 mg / mL DMSO-dissolved acridinium ester solution was added, and the mixture was thoroughly mixed using a vortex shaker, and reacted at 25°C in the dark for 60 min; Termination after labeling: add 6.67 μL of 0.2 M / L lysine solution dissolved in pure water, mix thoroughly with a vortex shaker, and terminate at 25°C in the dark for 30 min; Post-termination purification: add 1000 μL washed Protein A liquid, incubate at 25°C in the dark with shaking for 15 min; add the mixed liquid of incubated antibody and Protein A to the purification empty column, add 1000 μL pH6.5 1X PBS to the centrifuge tube to wash the centrifuge tube, and then add the liquid to the purification empty column, repeat 3 times; purify at a speed of 1000 μL / min with a peristaltic pump for 1 h; Elution after purification: drain the liquid in the purification column, add 6000 μL pH 3.0 0.1M / L Tris-HCl elution solution to the column, incubate at 25°C in the dark for 15 minutes; elute the antibody by gravity; Final preservation: Add an equal amount of 2X acridinium ester preservation solution to the eluted liquid, mix quickly and thoroughly using a vortex shaker, and store in a refrigerator at 2-8°C.

[0035] It should be noted that the performance evaluation of the above-mentioned TAT complex assay kit is as follows: A. Standard curve preparation: The TAT complex determination blocking reagent described in Example 1 was diluted with a standard diluent to be configured into calibration solution SO-S6 of different concentrations, with concentrations of 3.75ng / mL, 7.5g / mL, 15ng / mL, 30ng / mL, 60ng / mL, and 120ng / mL, respectively, and stored at -20°C for standby use. The TAT complex determination kit of the present embodiment is then used to detect the calibration product, and the luminescence intensity values ​​corresponding to each calibration product are read respectively. The concentration is used as the abscissa and the luminescence intensity is used as the ordinate to fit the standard curve.

[0036] B, Linear: The high-concentration sample is the basic bovine plasma sample with added antigen or the calibration diluent. The concentration of the calibration diluent is ≤ the blank limit. The real high-value serum sample should be used as much as possible. The bovine plasma sample or the calibration diluent is used as the low-concentration sample. The high-value sample close to the upper limit of the linear range is diluted to several concentrations according to a certain ratio. The table can be prepared according to the linear sample. The low-value concentration sample must be close to the lower limit of the linear range. The sample of each concentration is tested 3-5 times and the average value is calculated. The concentration value of the analyte is used as the horizontal axis and the average value of the test result is used as the vertical axis. The least squares method is used for linear fitting. The correlation coefficient r is calculated within the linear range, and the acceptance standard is the correlation coefficient r>0.99.

[0037] As shown in the following table, samples with different protein concentrations were tested using a kit without a blocking agent, a kit containing a blocking agent, and the TAT complex detection kit of this embodiment, and the results were obtained. Figure 2 The test results are shown.

[0038]

[0039] C, minimum detection limit: Use the zero-concentration calibrant dilution as the sample for detection, repeat the measurement 20 times, and obtain the light signal value of the 20 measurement results. Calculate the average value M and standard deviation SD to obtain M+2SD. Perform two-point regression fitting on the concentration and light signal value results between the zero-concentration enterprise linear reference and the adjacent concentration calibrant to obtain a linear equation. Substitute the light signal value of M+2SD into the equation to find the corresponding concentration value, which is the minimum detection limit.

[0040] D, accuracy: Prepare samples with high and low concentrations using the thrombin-antithrombin m complex, repeat the measurement three times, calculate the average value, record it as the relative deviation of M from the labeled value, and calculate the relative deviation B of the measured concentration according to formula (1). Alternatively, use the reagent to be evaluated to test samples with high and low concentrations that have been determined by the reference method, repeat the measurement three times for each concentration sample, take the average value of the test results, and calculate its relative deviation from the labeled value. Acceptance standard: relative deviation within ±10%.

[0041] Formula (1): B = (MT) / TX100%; B: relative deviation; M: average value of measured concentration; T: indicated value.

[0042] E, precision: E1, Repeatability evaluation: The samples at 2 to 3 concentration levels were tested 10 times each, and the mean value M and standard deviation SD of the 10 measurement results were calculated. The coefficient of variation CV was obtained according to formula (2).

[0043] Formula (2): CV = SD / M×100%; CV: coefficient of variation; SD: standard deviation of 10 measurement results; M: mean value of 10 measurement results.

[0044] E2, batch-to-batch precision: The precision of the results of measurements of the same test sample by the same operator on the same instrument, using the same method and the same type and batch of reagents over a period of time, generally one month or 20 working days.

[0045] F, interfering substances: Weigh the interfering substance to be tested. Prepare a stock solution of a certain concentration of the interfering substance. Add bilirubin, triglyceride, hemoglobin and total protein stock solutions to the high and low value plasmas used for interference according to the calibrated concentrations. Prepare high concentration interfering substance plasma samples containing bilirubin, triglyceride, hemoglobin and total protein at the concentrations required by the specifications. The control plasma sample is the one without added interfering substance. The added volume is controlled within 1 / 20 to avoid the matrix effect of the solvent. Test the high concentration interfering substance sample and the control plasma sample 2-3 times respectively. The mean result of the high concentration interfering substance sample test is recorded as M. The mean result of the control plasma sample test is recorded as T, and the relative deviation B of the measured concentration is calculated according to formula (4).

[0046] Formula (4): B = (MT) / TX100%; where: Add the interfering substance to the high and low value plasma B: relative deviation; M: mean of the analyte concentrations in high and low concentration interferent samples; T: mean of the analyte concentrations in control plasma samples.

[0047] Kit analysis performance evaluation results:

[0048] Regarding the clinical performance of the TAT complex assay kit of this example: 151 clinical plasma samples were simultaneously tested using the TAT complex assay kit of this example and the existing kit.

[0049] The detection method of the TAT complex assay kit of this embodiment is as follows: Before starting the test, check whether the instrument has completed the self-test successfully and prepare the materials required for the test. The basic test operation is as follows: Load consumables, including consumable boxes, reaction cup boxes, substrate liquid bottles and reagent boats. When loading the reagent boat, you should gently flip the reagent boat and manually rotate the bottom disc of the chamber containing the magnetic balls before tearing the seal until the magnetic particles are completely suspended. If the magnetic particles are still attached to the bottom of the reagent bottle after multiple flips, the bottle of reagent cannot be used, please contact customer service. Do not flip the reagent boat with the seal torn.

[0050] Load no less than 250µL of sample. Enter the sample application interface and enter relevant information.

[0051] Touch the Start button, select the test sample, test items (including the test dilution factor), and enter the number of repetitions.

[0052] The instrument reads the sample information and test items, and automatically performs sample testing. The instrument draws 20µL of the sample to be tested and 50µL of magnetic bead working solution (R1) into the reaction cup, incubates at 37℃ (±0.3℃) for 5 minutes, then washes, adds 50µL of acridinium ester marker (R2), incubates at 37℃ (±0.3℃) for 5 minutes, then washes, adds the substrate solution for the fully automatic immunoassay system, and measures the luminescent signal. Enter the result interface to view the test results.

[0053] The existing test kit is used to perform concentration detection on the above-mentioned samples.

[0054] The detection concentration obtained by the TAT complex assay kit of this example was analyzed and compared with the detection result concentration of the restricted kit, and the clinical relevance results were as follows: Figure 3 As shown, the clinical correlation is R2=0.902, and the result meets the acceptance standard, acceptance standard: R2>0.9, indicating that the kit of the present invention has a good correlation with the existing kit.

[0055] The detection concentration obtained by the TAT complex assay kit of this embodiment and the concentration of the existing kit detection result are statistically analyzed for the positive and negative coincidence rate, according to the reference interval or clinical judgment value. The positive and negative coincidence rate calculation table is as follows:

[0056] The final test results are as follows:

[0057] The above experiments show that the present application realizes the quantitative detection of TAT in samples, and has the advantages of high detection sensitivity, high accuracy, good stability, simple operation, high throughput, etc., which can meet the needs of clinical TAT quantitative detection. Compared with the limited detection kits at home and abroad, it has higher detection stability and lower detection cost.

Claims

1. A method for determining a TAT complex, characterized in that: Prothrombin is blocked by a blocking antibody around the 274th amino acid of prothrombin, and thrombin is detected by a detection antibody acting on the N-terminus of thrombin, so that the detection antibody cannot bind to prothrombin.

2. A TAT complex assay blocking antibody, used in a TAT complex assay method according to claim 1, characterized in that: The blocking antibody blocks prothrombin around amino acid position 274.

3. A TAT complex assay detection antibody, used in a TAT complex assay method according to claim 1, characterized in that: The detection antibody acts on the N-terminus of thrombin.

4. A TAT complex assay kit, implemented using the TAT complex assay method of claim 1, characterized in that: Includes detection antibodies acting on the N-terminus of thrombin.

5. A method for preparing a TAT complex assay kit, for preparing the TAT complex assay kit according to claim 4, characterized in that: The prepared TAT complex assay reagent is placed in an assay kit to obtain a TAT complex assay kit, wherein the TAT complex assay kit contains a blocking antibody. The preparation of the TAT complex assay reagent includes: Step 1: Prepare magnetic bead coating buffer; Step 2: Prepare magnetic bead cleaning solution; Step 3: Prepare magnetic bead blocking solution; Step 4: Prepare magnetic bead storage solution; Step 5: prepare magnetic microspheres for first washing, activation, second washing, coating, third washing, blocking, and storage after fourth washing, using magnetic bead washing solution for washing, magnetic bead coating buffer for activation, and magnetic bead blocking solution for blocking. When storing, store the magnetic microspheres in magnetic bead storage solution; Step 6: Use the detection antibody to carry out acridinium ester labeling, and then terminate, purify, elute, and finally store to obtain the TAT complex determination reagent.

6. The method for preparing a TAT complex assay kit according to claim 5, characterized in that: The raw materials of the coating buffer include MES, the raw materials of the magnetic bead washing solution include Tris, sodium chloride and Tween-20, the raw materials of the magnetic bead blocking solution include Tris, sodium chloride and BSA, and the raw materials of the magnetic bead preservation solution include Hepes, sodium chloride, BSA, sucrose, Tween-20 and Proclin300.

7. The method for preparing a TAT complex assay kit according to claim 5, characterized in that: During activation, magnetic bead coating buffer was added to the washed magnetic microspheres, NHS solution dissolved in pure water was added after thorough mixing, and the mixture was resuspended using a vortex shaker. EDC solution dissolved in pure water was added after thorough mixing, and the mixture was resuspended using a vortex shaker. After thorough mixing, the mixture was shaken and incubated.

8. The method for preparing a TAT complex assay kit according to claim 5, characterized in that: During coating, the detection antibody solution and magnetic bead coating buffer were added to the washed magnetic microspheres, resuspended by vortex shaking instrument, quickly mixed and then incubated by shaking.

9. The method for preparing a TAT complex assay kit according to claim 5, characterized in that: During blocking, add BSA diluted with pure water to the washed magnetic microspheres, resuspend them using a vortex shaker, mix them thoroughly, and then shake and incubate them.

10. The method for preparing a TAT complex assay kit according to claim 5, characterized in that: Acridinium ester labels include: Step 1: Prepare a labeling buffer, the raw materials of which include disodium hydrogen phosphate and sodium chloride; Step 2: Prepare a stop solution, the raw materials for preparing the stop solution include lysine; Step 3: prepare acridinium ester preservation solution, the raw materials of which include MES, sodium chloride, BSA, sucrose, glycerol, Tween-20 and Proclin300; Step 4: labeling with acridinium ester using detection antibody and labeling buffer, and then performing termination, purification, elution, and finally storage to obtain TAT complex assay reagent. When terminating, add termination solution, and when eluting, add acridinium ester storage solution.

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