Method for measuring thrombin-antithrombin III complex, blocking antibody, detection antibody, and kit

Abstract

The invention discloses a thrombin-antithrombin III compound determination method, a blocking antibody, a detection antibody and a kit, the blocking antibody blocks the vicinity of the 274-site residue of the prothrombin nitrogen terminal, and the detection antibody is used as a capture antibody of a TAT compound and specially aims at the effect of thrombin close to the thrombin N terminal. When the closing antibody near the position behind the 274-site residue of the prothrombin nitrogen terminal is combined with the prothrombin antibody, the closing antibody can effectively prevent the detection antibody at the N terminal of the thrombin from combining with the prothrombin, but because the closing antibody near the position behind the 274-site residue of the prothrombin nitrogen terminal cannot combine with the thrombin, the closing antibody can effectively prevent the detection antibody at the N terminal of the thrombin from combining with the prothrombin. 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 thrombin-antithrombin III complex determination, and specifically to a thrombin-antithrombin III 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 (AT) to form the thrombin-antithrombin complex (TAT), which is a molecular marker reflecting thrombin generation. 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 a protease of the coagulation system stimulated by Na+ ions and is a key enzyme involved in the coagulation cascade reaction. After an organism is injured, thrombin rapidly 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 connected by covalent disulfide bonds and composed of two subunits, A and B. The molecular weight is 37k D. The A chain contains 36 residues and is located on the back of the molecule. It stabilizes the complete functionality of thrombin and is called the light chain; the B chain contains 259 residues and has the activity and functional domain of the enzyme, which 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 related products on the market include the thrombin-antithrombin III complex detection kit produced by Sysmex Medical Electronics (Shanghai) Co., Ltd., and the domestic marketed products are mainly the thrombin-antithrombin III complex detection kit mainly produced by Guangzhou Wondfo Biotechnology Co., Ltd., all of which are suitable for chemiluminescence method, 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:

[0006] 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;

[0007] 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.

[0008] 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.

[0009] Application Contents

[0010] The purpose of the present application is to provide a thrombin-antithrombin III 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.

[0011] To achieve the above-mentioned purpose, the present application provides the following technical solutions: a method for measuring thrombin-antithrombin III complex, using a blocking antibody to block prothrombin before and after the amino acid 274 of prothrombin, and using a detection antibody acting on the N-terminus of thrombin to detect thrombin, so that the detection antibody cannot bind to prothrombin.

[0012] 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.

[0013] A blocking antibody is used to measure the thrombin-antithrombin III complex. 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 the detection accuracy.

[0014] A thrombin-antithrombin III complex detection antibody, the detection antibody acts on the N-terminus of thrombin.

[0015] A thrombin-antithrombin III complex detection kit comprises a detection antibody acting on the N-terminus of thrombin.

[0016] Compared with the prior art, the beneficial effects of this application are:

[0017] (1) The thrombin-antithrombin III complex assay 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;

[0018] (2) The blocking antibody for the thrombin-antithrombin III 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

[0019] Figure 1A schematic diagram of the blocking antibody action site for the thrombin-antithrombin III complex assay of the present application;

[0020] Figure 2 A comparison chart of the test results of a thrombin-antithrombin III complex assay kit proposed in the present application and existing kits;

[0021] Figure 3 This is a clinical comparison chart between a thrombin-antithrombin III complex assay kit proposed in this application and existing kits. DETAILED DESCRIPTION

[0022] 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.

[0023] Embodiment 1

[0024] A method for measuring thrombin-antithrombin III complex 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 high concentration of prothrombin in plasma from interfering with the TAT complex detection process.

[0025] Embodiment 2

[0026] A blocking antibody used in the thrombin-antithrombin III complex assay method of Example 1, wherein the blocking antibody blocks prothrombin around the 274th amino acid of prothrombin.

[0027] Embodiment 3

[0028] A detection antibody used in the thrombin-antithrombin III complex determination method of Example 1, wherein the detection antibody acts on the N-terminus of thrombin.

[0029] Embodiment 4

[0030] A thrombin-antithrombin III complex assay kit comprises the detection antibody acting on the N-terminus of thrombin of Example 3.

[0031] The preparation method of the thrombin-antithrombin III complex assay kit of this embodiment is as follows: first, prepare magnetic beads for coating, taking coating 10 mg of magnetic beads as an example,

[0032] The first step is to prepare 1000ml of coating buffer. The specific formula and conditions are as follows:

[0033]

[0034] The second step is to prepare 1000ml of magnetic bead cleaning solution. The specific formula and conditions are as shown in the following table.

[0035]

[0036]

[0037] The third step is to prepare 1000ml of magnetic bead blocking solution. The specific formula and conditions are as shown in the following table.

[0038]

[0039] Step 4: Prepare 1000ml of magnetic bead preservation solution. The specific formula and conditions are as shown in the following table.

[0040]

[0041] 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 oscillator, mix thoroughly, place on a magnetic rack, 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 cleaning: 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 oscillator, and mix quickly and thoroughly; finally, add 100 μL of 10 mg / mL EDC solution (dissolved in pure water), resuspend with a vortex oscillator, and mix quickly and thoroughly; incubate at 25°C with shaking for 30 minutes;

[0042] 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;

[0043] 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;

[0044] 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;

[0045] 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;

[0046] 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;

[0047] 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.

[0048] Next, we will perform acridinium ester labeling, taking 0.2 mg of antibody as an example.

[0049] First, prepare 1000 ml of labeling buffer. The specific formula and conditions are as shown in the following table.

[0050]

[0051] Prepare 1000ml of stop solution. The specific formula and conditions are as shown in the following table.

[0052]

[0053] Prepare 1000 ml of acridinium ester preservation solution. The specific formula and conditions are as shown in the following table.

[0054]

[0055] 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. The mixture was reacted at 25°C in the dark for 60 min.

[0056] 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 minutes;

[0057] 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 pH6.51X 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;

[0058] Elution after purification: drain the liquid in the purification column, add 6000 μL pH 3.00.1M / L Tris-HCl elution solution to the column, incubate at 25°C in the dark for 15 minutes; elute the antibody by gravity;

[0059] 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.

[0060] It should be noted that the performance evaluation of the above-mentioned thrombin-antithrombin III complex assay kit is as follows:

[0061] A. Standard curve preparation:

[0062] The blocking reagent for the thrombin-antithrombin III complex assay described in Example 1 was diluted with a standard diluent to prepare calibration solution SO-S6 of different concentrations, with concentrations of 3.75 ng / mL, 7.5 g / mL, 15 ng / mL, 30 ng / mL, 60 ng / mL, and 120 ng / mL, respectively, and stored at -20 ° C for use. Then, the thrombin-antithrombin III complex assay kit of this embodiment was used to detect the calibrator, and the luminescence intensity values ​​corresponding to each calibrator were read respectively. The standard curve was obtained by fitting with concentration as the horizontal axis and luminescence intensity as the vertical axis.

[0063] B, Linear:

[0064] The high-concentration sample is the basic bovine plasma sample or the calibration diluent with added antigen. The concentration of the calibration diluent is ≤ the blank limit. Try to use the real high-value serum sample. The bovine plasma sample or the calibration diluent is 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. Repeat the test 3-5 times for each concentration of the sample, and calculate the average value. 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, and the least squares method is used for linear fitting. And calculate the correlation coefficient r within the linear range, and the acceptance standard is the correlation coefficient r>0.99.

[0065] 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 thrombin-antithrombin III complex detection kit of this embodiment, and the results were obtained. Figure 2 The test results are shown.

[0066] Protein concentration (ng / mL) Kit without blocking agent Kit with blocking agent This application kit 3.75 11465 27678 19901 7.5 21351 51504 40173 15 63607 91923 77964 30 75489 180499 149920 60 106384 376214 339416 120 137107 715868 605404

[0067] C, minimum detection limit:

[0068] 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 based 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.

[0069] D, accuracy:

[0070] 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%.

[0071] Formula (1): B = (MT) / TX100%; B: relative deviation; M: average value of measured concentration; T: indicated value.

[0072] E, precision:

[0073] E1, Repeatability evaluation:

[0074] 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).

[0075] Formula (2): CV = SD / M × 100%; CV: coefficient of variation; SD: standard deviation of 10 measurement results; M: average value of 10 measurement results.

[0076] E2, batch-to-batch precision:

[0077] The precision of the results of the same test sample measured 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. F, interfering substances:

[0078] 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).

[0079] Formula (4): B = (MT) / TX100%; where: when the interfering substance is added to the high and low value plasma, B: relative deviation; M: mean value of the concentration of the analyte in the high and low concentration interfering substance samples; T: mean value of the concentration of the analyte in the control plasma samples.

[0080] Kit analysis performance evaluation results:

[0081]

[0082] Regarding the clinical performance of the thrombin-antithrombin III complex assay kit of this example:

[0083] 151 clinical plasma samples were simultaneously tested using the thrombin-antithrombin III complex assay kit of this example and the kit of Guangzhou Wondfo Biotechnology Co., Ltd.

[0084] The detection method of the thrombin-antithrombin III complex assay kit of this embodiment is as follows: Before the test begins, check whether the instrument has successfully completed the self-test and prepare the materials required for the test.

[0085] The basic test operations are as follows:

[0086] 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.

[0087] Load no less than 250μL of sample. Enter the sample application interface and enter relevant information.

[0088] Touch the start button, select the test sample, test items (including test dilution factor), and enter the number of repetitions.

[0089] The instrument reads the sample information and test items, and automatically performs sample testing. ⑤ 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. ⑥ Enter the result interface to view the test results.

[0090] The concentration of the above-mentioned samples was also tested using the kit produced by Guangzhou Wondfo Biotech Co., Ltd.

[0091] The detection concentration obtained by the thrombin-antithrombin III complex assay kit of this example was analyzed and compared with the detection result concentration of the kit of Guangzhou Wondfo Biotechnology Co., Ltd. The clinical relevance results are as follows Figure 3 As shown, the clinical correlation is R2=0.902, and the result meets the acceptance criteria, acceptance criteria: R2>0.9, indicating that the kit of the present invention has a good correlation with the kit of Guangzhou Wondfo Biotech Co., Ltd.

[0092] The detection concentration obtained by the thrombin-antithrombin III complex assay kit of this example and the detection result concentration of the kit of Guangzhou Wanfu Biotechnology Co., Ltd. were 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:

[0093]

[0094]

[0095] The final test results are as follows:

[0096]

[0097] 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 measuring thrombin-antithrombin III complex, characterized in that: The prothrombin is blocked by a blocking antibody around the 274th amino acid of the prothrombin, and the thrombin is detected by a detection antibody acting on the N-terminus of the thrombin, so that the detection antibody cannot bind to the prothrombin.

2. A thrombin-antithrombin III complex assay blocking antibody, characterized in that: The blocking antibody blocks prothrombin around amino acid 274 of prothrombin.

3. A thrombin-antithrombin III complex assay detection antibody, characterized in that: The detection antibody acts on the N-terminus of thrombin.

4. A thrombin-antithrombin III complex assay kit, characterized in that: Includes detection antibodies acting on the N-terminus of thrombin.

Images