Method and kit for detecting ADAMTS13 activity and inhibitor

Abstract

The invention discloses a method for detecting the activity of ADAMTS13, a method for detecting an ADAMTS13 inhibitor and a kit. The method comprises the following steps: exposing a VWF restriction enzyme cutting site through urea by adopting an ELISA (Enzyme-Linked Immunosorbent Assay) combined dialysis technology, cutting VWF by utilizing ADAMTS13, separating large and small molecule VWF through dialysis, finally detecting the absorbance ratio of the VWF before and after dialysis through the ELISA, and calculating the activity or inhibitor state of ADAMTS13. The method provided by the invention has the advantages of strong specificity, high sensitivity and good repeatability, and is suitable for large-scale sample detection.

Description

Technical Field

[0001] This invention relates to the field of clinical testing technology, specifically to a method and kit for detecting ADAMTS13 activity and its inhibitors. Background Technology

[0002] von Willebrand factor lyase (ADAMTS13) belongs to the ADAMTS subfamily of metalloproteinases. It can specifically cleave large molecules of von Willebrand factor (VWF) in plasma, regulating the structure and function of VWF. Deficiency can lead to thrombotic thrombopurpura (TTP), causing microvascular thrombosis, resulting in consumptive thrombocytopenia, and dysfunction of organs such as the heart, brain, and kidneys.

[0003] VWF is a large polymeric glycoprotein in plasma, synthesized by megakaryocytes and endothelial cells. It not only acts as a bridge between platelets and damaged blood vessels in primary hemostasis but also indirectly affects the coagulation pathway by stabilizing coagulation factor VIII. Its function depends on its polymeric state; the larger the molecular weight, the stronger its binding affinity to collagen and platelets. ADAMTS13 regulates the adhesion of VWF polymers to subendothelial collagen and platelets by controlling the size of VWF polymers. It acts on the peptide bond between tyrosine (position 1605) and methionine (position 1606) within the VWF-A2 domain, cleaving the VWF polymer to achieve an appropriate size. This prevents VWF-induced excessive platelet aggregation and thrombus formation, thereby maintaining coagulation homeostasis. When plasma ADAMTS13 antigen or activity is reduced, VWF polymers accumulate in the blood, causing abnormal platelet aggregation and widespread microvascular thrombosis, thereby inducing TTP. Patients may present with some or all of the following symptoms: thrombocytopenia, hemolytic anemia, neurological abnormalities, renal insufficiency, and fever.

[0004] TTP is classified into two types: hereditary (cTTP) and immune (iTTP). Hereditary TTP is caused by gene mutations that reduce or eliminate ADAMTS13. Immune TTP is often caused by autoantibodies that inhibit ADAMTS13 activity or accelerate its clearance. Antibodies against ADAMTS13 are divided into two categories: inhibitory antibodies, which bind to ADAMTS13 and affect its activity; and non-inhibitory antibodies, which bind to ADAMTS13 and accelerate its clearance from the plasma. Homozygous or double heterozygous mutations in the ADAMTS13 gene can confirm hereditary TTP.

[0005] Currently, there are various methods for detecting ADAMTS13 activity, including VWF multimer analysis, quantitative immunoblotting, enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) technology.

[0006] The FRETS-VWF73 method is currently the most widely used and considered the "gold standard" method. This method works by using a synthetic fluorescent substrate (FRETS-VWF73) containing the specific cleavage sequence of ADAMTS13 within the A2 domain of VWF, labeled with a fluorescent donor and a quencher at both ends. When ADAMTS13 cleaves the substrate, the fluorescent and quencher groups separate, generating a fluorescent signal. The rate of change in fluorescence intensity over time directly reflects the enzyme activity of ADAMTS13. However, this method has the following drawbacks: 1) Requires specialized equipment: It relies on a fluorometer or a dedicated coagulation analyzer, resulting in high equipment costs. 2) Expensive substrate: The synthetic and labeled FRETS-VWF73 peptide is expensive. 3) Interference from fluorescent substances in plasma: Hyperbilirubinemia, hemolysis (red plasma), etc., can interfere with fluorescence readings, potentially leading to falsely low activity. 4) Insensitive to certain mutants: The substrate is a short peptide, which may not be able to completely mimic the conformation of the full-length VWF multimer. For some mutations that affect the binding of ADAMTS13 to the full-length VWF but not to the binding of small peptide substrates, false negatives (normal activity) may occur. Summary of the Invention

[0007] This invention provides an ELISA-based method for detecting ADAMTS13 activity and inhibitors using dialysis. By optimizing dialysis conditions, sample processing steps, and the detection process, the method improves the specificity, sensitivity, and accuracy of the detection, making it suitable for rapid detection of large batches of clinical samples. Urea is used to expose the active sites of large-molecule VWF in plasma, which are then cleaved into smaller molecules by ADAMTS13. Residual VWF after dialysis is detected to determine ADAMTS13 activity. The passage of cleaved small-molecule VWF through the dialysis membrane indicates ADAMTS13 activity; low activity means that large-molecule VWF (greater than 20000 kDa) cannot be cleaved and cannot pass through the dialysis membrane. The ratio of absorbance of dialysis-prepared residual VWF to that of predialysis-prepared residual VWF represents activity. The method exhibits high specificity and sensitivity.

[0008] A method for detecting ADAMTS13 activity includes the following steps: taking two equal plasma samples from the plasma to be tested; mixing one plasma sample with urea-containing dialysate and dialyzing at 37°C for 13-16 hours; diluting the dialyzed plasma sample and the other undialyzed plasma sample separately; and performing ELISA to determine the VWF content separately. The ADAMTS13 activity is calculated based on the absorbance ratio of the dialyzed to the undialyzed plasma sample.

[0009] Furthermore, ADAMTS13 activity = 100% - (absorbance value of dialysis-treated plasma sample / absorbance value of undialysis-treated plasma sample) × 100%.

[0010] Furthermore, the preparation method of the dialysate is as follows: 1) Prepare 5× concentrate: Weigh Tris, add deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl; add urea to the above solution, stir to dissolve completely, and make up to volume; finally add Tween 20 to prepare 5× concentrate; take the above 5× concentrate, dilute it according to the ratio, mix well and prepare 1× working solution.

[0011] Furthermore, during dialysis, the plasma sample is placed in the dialysis tube, which is then completely immersed in the dialysate.

[0012] Furthermore, the plasma to be tested is obtained by centrifugation after collecting sodium citrate-anticoagulated venous blood.

[0013] This invention provides a method for detecting ADAMTS13 inhibitors, comprising the following steps: mixing a plasma sample to be tested with plasma from a healthy person at a volume ratio of 9:1 and incubating at 37°C for 3 hours; separating two equal plasma samples from the mixed plasma; dialyzing one of the plasma samples at 37°C for 13-16 hours; diluting the dialyzed plasma sample with the other undialyzed plasma sample; performing ELISA to determine the VWF content; and determining the detection status of the inhibitor based on the absorbance ratio of the dialyzed to the undialyzed plasma sample.

[0014] If the ratio of the absorbance value of the dialyzed plasma sample to that of the undialyzed plasma sample is less than 10%, it is considered a positive inhibitor.

[0015] Furthermore, the preparation method of the dialysate is as follows: 1) Prepare 5× concentrate: Weigh Tris, add deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl; add urea to the above solution, stir to dissolve completely, and make up to volume; finally add Tween 20 to prepare 5× concentrate; take the above 5× concentrate, dilute it according to the ratio, mix well and prepare 1× working solution.

[0016] Furthermore, during dialysis, the plasma sample is placed in the dialysis tube, which is then completely immersed in the dialysate.

[0017] Furthermore, the plasma to be tested is obtained by centrifugation after collecting sodium citrate-anticoagulated venous blood.

[0018] The present invention also provides a kit for detecting ADAMTS13 activity, the kit being used to implement a method for detecting ADAMTS13 activity, comprising: dialysis buffer; VWF ELISA assay reagent; rabbit anti-human VWF polyclonal antibody; HRP-labeled secondary antibody; substrate solution and stop solution.

[0019] Furthermore, the dialysate contains Tris buffer, urea, and Tween 20, with a pH of 8.3.

[0020] The kit includes the following steps for detecting ADAMTS13 activity: two equal plasma samples are aliquoted from the plasma to be tested; one plasma sample is mixed with urea-containing dialysate and dialyzed at 37°C for 13-16 hours; the dialyzed plasma sample is diluted with the other undialyzed plasma sample; and the VWF content is determined by ELISA. The ADAMTS13 activity is calculated based on the absorbance ratio of the dialyzed to the undialyzed plasma sample.

[0021] The preparation method of the dialysate is as follows: 1) Prepare 5× concentrate: Weigh Tris, add deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl; add urea to the above solution, stir to dissolve, and make up to volume; finally add Tween 20 to prepare 5× concentrate; take the above 5× concentrate, dilute it according to the ratio, mix well and prepare 1× working solution.

[0022] During dialysis, the plasma sample is placed in the dialysis tube, ensuring the tube is completely submerged in the dialysate.

[0023] The present invention also provides a kit for detecting ADAMTS13 inhibitors, the kit being used to implement a method for detecting ADAMTS13 inhibitors, comprising: dialysis buffer; VWF ELISA assay reagent; rabbit anti-human VWF polyclonal antibody; HRP-labeled secondary antibody; substrate solution and stop solution.

[0024] Furthermore, the dialysate contains Tris buffer, urea, and Tween 20, with a pH of 8.3.

[0025] The kit, used for the method of detecting ADAMTS13 inhibitors, includes the following steps: mixing the plasma sample to be tested with healthy human plasma at a volume ratio of 9:1 and incubating at 37°C for 3 hours; separating two equal plasma samples from the mixed plasma; dialyzing one plasma sample at 37°C for 13-16 hours; diluting the dialyzed plasma sample with the other undialyzed plasma sample; performing ELISA to determine the VWF content; and determining the detection status of the inhibitor based on the absorbance ratio of the dialyzed to the undialyzed plasma sample.

[0026] The preparation method of the dialysate is as follows: 1) Prepare 5× concentrate: Weigh Tris, add deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl; add urea to the above solution, stir to dissolve, and make up to volume; finally add Tween 20 to prepare 5× concentrate; take the above 5× concentrate, dilute it according to the ratio, mix well and prepare 1× working solution.

[0027] During dialysis, the plasma sample is placed in the dialysis tube, ensuring the tube is completely submerged in the dialysate.

[0028] The beneficial effects of this invention are:

[0029] 1. Methodological Integration and Innovation: By combining ELISA technology with dialysis technology, VWF restriction sites are exposed by urea, VWF is cleaved by ADAMTS13, and VWF molecules of different sizes are separated by dialysis. Finally, the VWF content before and after dialysis is detected by ELISA, which achieves efficient detection of ADAMTS13 activity or inhibitors.

[0030] 2. Dialysis buffer formulation optimization: A specific dialysis buffer formulation (containing Tris buffer, urea and Tween 20, pH 8.3) was used to optimize the exposure conditions of VWF restriction sites, thereby improving the specificity and sensitivity of the detection.

[0031] 3. Improved dialysis method: The innovative method of "complete immersion of dialysis tube" is adopted, which results in more complete dialysis, less change in sample volume, and more stable test results compared to partial immersion.

[0032] 4. Innovative method for detecting inhibitors: A detection process based on "mixing and incubating test plasma with healthy human plasma → dialysis → ELISA detection" is proposed. The state of inhibitors is determined by judging whether the VWF content changes before and after dialysis. The method is simple and reliable.

[0033] 5. Integrated kit design: A complete kit is provided, including dialysis solution, ELISA detection reagent, antibody, substrate, etc., which facilitates standardized operation and large-scale clinical application. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments. Example 1: Preparation of Dialysis Solution

[0035] Preparation of dialysis solution: 1. Prepare 5 x 100mL concentrated solutions: (1) Weigh Tris according to Table 1, add an appropriate amount of deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl.

[0036] (2) Add urea to the dissolved Tris buffer and stir thoroughly to dissolve. Urea absorbs heat when it dissolves and the temperature drops. If the dissolution is incomplete, cool it to room temperature and continue stirring. Finally, add an appropriate amount of deionized water to make up to 100 ml.

[0037] (3) After adjusting the volume, add Tween20, mix well, and obtain 5×100ml concentrated solution.

[0038] 2. Dilute to 1× working solution: Take the above 5× concentrate, dilute it at a ratio of 1:4, mix well to obtain 1× working solution, which can be used for subsequent detection reactions.

[0039] Table 1

[0040] Example 2 ADAMTS13 Activity Detection Sample preparation: Collect venous blood anticoagulated with sodium citrate, centrifuge to separate plasma, and obtain the plasma to be tested.

[0041] Sample processing: Two equal plasma samples, 50 μL each, were taken from the plasma to be tested and labeled A and B. Sample A was stored at room temperature for later use; sample B was placed at 37°C and dialyzed completely in dialysis buffer for 13-16 h.

[0042] 3. ELISA test: 1) Use PBS buffer to bring A to the same volume as the dialyzed B, then dilute A and B 20 times at the same time.

[0043] 2) Set up blank wells, sample wells, and PBS buffer wells. Add 100 μL of A and B to each sample well for replicates; do not add samples to the blank wells. Affix the sealing film and incubate at 37°C for 2 hours. Discard the liquid in the wells and agitate. Add 100 μL of diluted HRP-labeled antibody to each well, cover with a new plate, and incubate at 37°C for 1 hour. Discard the liquid in the wells, agitate, and wash the plate 5 times. Immerse for 2 minutes each time, adding 200 μL per well, and agitate. Add 90 μL of substrate solution to each well and incubate at 37°C in the dark for 20 minutes to stop the reaction. Add 50 μL of stop solution to each well to terminate the reaction. The substrate solution and stop solution are products of Huamei Biotechnology.

[0044] Within 5 minutes after the reaction was terminated, the OD value of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0045] The absorbance value (OD value) obtained by adding sample A to the sample well is the absorbance value of the undialyzed plasma sample, and the absorbance value obtained by adding sample B to the sample well is the absorbance value (OD value) of the dialyzed plasma sample.

[0046] Activity calculation: ADAMTS13 activity = 100% - (absorbance value of dialysis-treated plasma sample / absorbance value of undialysis-treated plasma sample) × 100%. Example 3: Detection of ADAMTS13 Inhibitor

[0047] 1. Sample preparation: Collect venous blood anticoagulated with sodium citrate, centrifuge to separate plasma, and obtain the plasma to be tested.

[0048] 2. Sample processing: Mix the plasma to be tested with plasma from healthy individuals at a volume ratio of 9:1, and incubate the mixture at 37°C for 3 hours. Then, aliquot two equal plasma samples, 50 μL each, and label them C and D, respectively. Sample C is stored at room temperature for later use; sample D is placed at 37°C and dialyzed completely in dialysis buffer for 13-16 hours.

[0049] 3. ELISA detection: The detection steps for C and D are the same as those for A and B in Example 2.

[0050] The absorbance value (OD value) obtained from the sample well with C is the absorbance value (OD value) of the undialyzed plasma sample, and the absorbance value (OD value) obtained from the sample well with D is the absorbance value (OD value) of the dialyzed plasma sample.

[0051] 4. Result Interpretation: If the ratio of the absorbance value of the dialysis-treated plasma sample to that of the undialysis-treated plasma sample is less than 10%, it is judged as positive for the inhibitor; otherwise, it is judged as negative for the inhibitor. Example 4: Validation of ADAMTS13 activity detection method

[0052] Forty-four samples were tested, of which samples 1-28, 30, and 32-43 were normal samples, and samples 29, 31, and 44 were ITP samples.

[0053] The 44 samples were tested according to the steps in Example 2, and the results are shown in Table 2 below.

[0054] The average ADAMTS13 activity was 65.23% ± 14.60%, and all inhibitors were negative, which was in line with expectations. The activity in the three ITP samples (29, 31, and 44) ​​was normal (ADAMTS13 activity less than 10% indicates thrombotic thrombocytopenic purpura (TTP)), further validating the good specificity of this method.

[0055] Table 2

[0056]

[0057]

[0058] Example 5: Validation of the ADAMTS13 inhibitor detection method A total of 20 normal human samples were tested for the inhibitor, and the results are shown in Table 3. All ADAMTS13 inhibitors were negative, which is basically in line with the experimental expectations.

[0059] Table 3

[0060] Example 6: Advantages of the dialysis method This embodiment is used to verify the impact of two dialysis methods on the test results.

[0061] The same plasma sample was taken and dialysis was performed in two ways (partial immersion of the dialysis tube and complete immersion of the dialysis tube). Each method was repeated 4 times. Each test was performed according to the steps in Example 2. Except for the immersion method of the dialysis tube, the other operation steps and conditions were kept the same.

[0062] Based on the test results in Tables 4 and 5, it was found that the method of completely immersing the dialysis tube resulted in more complete dialysis, greater sample volume variation, and more stable test results.

[0063] Table 4: Partial Immersion of Dialysis Tubes

[0064] Table 5: Dialysis tubing completely submerged .

Claims

1. A method for detecting ADAMTS13 activity, characterized in that, Includes the following steps: Two equal plasma samples were separated from the plasma to be tested; one plasma sample was mixed with urea-containing dialysate and dialyzed at 37°C for 13-16 hours; the dialyzed plasma sample was then diluted with the other undialyzed plasma sample. The VWF content was determined by ELISA. The ADAMTS13 activity was calculated as follows: 100% - (absorbance value of dialyzed plasma sample / absorbance value of undialyzed plasma sample) × 100% based on the absorbance ratio of dialyzed to undialyzed plasma sample.

2. The method according to claim 1, characterized in that, The preparation method of the dialysate is as follows: 1) Prepare 5× concentrate: Weigh Tris, add deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl; add urea to the above solution, stir to dissolve, and make up to volume; finally add Tween 20 to prepare 5× concentrate; take the above 5× concentrate, dilute it according to the ratio, mix well and prepare 1× working solution.

3. The method according to claim 1, characterized in that, During dialysis, the plasma sample is placed in the dialysis tube, ensuring the tube is completely submerged in the dialysate.

4. A method for detecting ADAMTS13 inhibitors, characterized in that, Includes the following steps: Mix the plasma sample to be tested with plasma from healthy individuals at a volume ratio of 9:1 and incubate at 37°C for 3 hours. Separate two equal plasma samples from the mixed plasma. Dialyze one of the plasma samples at 37°C for 13-16 hours. Dilute the dialyzed plasma sample with the other undialyzed plasma sample. Perform ELISA to determine the VWF content. If the ratio of the absorbance value of the dialyzed plasma sample to that of the undialyzed plasma sample is less than 10%, it is considered a positive inhibitor.

5. The method according to claim 4, characterized in that, The preparation method of the dialysate is as follows: 1) Prepare 5× concentrate: Weigh Tris, add deionized water to dissolve it completely, and adjust the pH to 8.3 with dilute HCl; add urea to the above solution, stir to dissolve, and make up to volume; finally add Tween 20 to prepare 5× concentrate; take the above 5× concentrate, dilute it according to the ratio, mix well and prepare 1× working solution.

6. The method according to claim 4, characterized in that, During dialysis, the plasma sample is placed in the dialysis tube, ensuring the tube is completely submerged in the dialysate.

7. A kit for detecting ADAMTS13 activity, characterized in that, The kit is used to implement the method for detecting ADAMTS13 activity as described in claims 1-3, and includes: dialysate; VWF ELISA assay reagent; rabbit anti-human VWF polyclonal antibody; HRP-labeled secondary antibody; substrate solution and stop solution.

8. The reagent kit according to claim 7, characterized in that, The dialysate contains Tris buffer, urea, and Tween 20, with a pH of 8.

3.

9. A kit for detecting ADAMTS13 inhibitors, characterized in that, The kit is used to implement the method for detecting ADAMTS13 inhibitors as described in claims 4-6, and includes: dialysate; VWF ELISA detection reagent; rabbit anti-human VWF polyclonal antibody; HRP-labeled secondary antibody; substrate solution and stop solution.

10. The reagent kit according to claim 9, characterized in that, The dialysate contains Tris buffer, urea, and Tween 20, with a pH of 8.3.

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