Antibody for ADAMTS-13 detection and application thereof

Abstract

The invention relates to the field of antibodies, in particular to an antibody for detecting ADAMTS-13 and application of the antibody. According to the ADAMTS13 monoclonal antibody provided by the invention, a light chain sequence of the monoclonal antibody comprises CDR1 as shown in SEQ ID NO.3, CDR2 as shown in SEQ ID NO.4 and CDR3 as shown in SEQ ID NO.5; the heavy chain sequence comprises a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8. In addition, the invention further provides a detection kit containing the monoclonal antibody, and the detection kit can effectively detect whether ADAMTS13 exists or not and / or the content of ADAMTS13 in a biological sample.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and specifically, the present invention relates to an antibody for detecting ADAMTS-13 and an application thereof. Background Art

[0002] Von Willebrand factor-cleaving protease (vWF-CP), also known as A disintegrin-like and metalloprotease with thrombospondin-1 motifs, 13th member of the family, ADAMTS13, can cleave von Willebrand factor (vWF) and is a metalloprotease that regulates the structure and function of vWF. ADAMTS13 can inhibit the adhesion of platelets to endothelial cells and subendothelial collagen by degrading vWF polymers, thereby delaying thrombosis.

[0003] Accurate measurement of ADAMTS13 antigen levels is crucial for assessing an individual's risk of thrombosis. The ratio of vWF / ADAMTS13 can predict a patient's risk of thrombotic vessels, as well as conduct a risk assessment of the patient's recovery after treatment to promptly eliminate the risk of thrombosis. At the same time, the detection of ADAMTS13 antigen levels together with the detection of ADAMTS13 autoantibodies and ADAMTS13 enzyme activity can assist in the diagnosis of thrombocytopenic purpura (TPP).

[0004] The most mature ADAMTS13 antigen detection methodology is mainly ELISA, which has the disadvantages of long detection time, complex operation, poor repeatability and specificity. Therefore, there is a need for a detection method with high sensitivity, simple operation, high degree of automation and low cost. Summary of the invention

[0005] Based on the defects in the prior art, the present invention provides an antibody for detecting ADAMTS-13 and its application.

[0006] First, in the first aspect, the present invention provides an anti-ADAMTS13 monoclonal antibody, which comprises a light chain sequence such as CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:5, and a heavy chain sequence such as CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8.

[0007] In one embodiment, the monoclonal antibody comprises the light chain variable region amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region amino acid sequence shown in SEQ ID NO.2.

[0008] In one embodiment, the monoclonal antibody further comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian antibody; optionally, the heavy chain constant region and the light chain constant region are derived from at least one of mouse, human, primate, bovine, horse, dairy cow, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink antibodies or their mutants.

[0009] In a preferred embodiment, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ or λ; optionally, the heavy chain constant region and the light chain constant region are both derived from murine antibodies or mutants thereof; optionally, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0010] In one embodiment, the antibody belongs to the IgG1 subclass, and its heavy chain constant region and light chain constant region have amino acid sequences characteristic of natural human IgG1 antibodies.

[0011] In a preferred embodiment, the heavy chain variable region nucleotide sequence of the monoclonal antibody is shown as SEQ ID NO:10, and the light chain variable region nucleotide sequence is shown as SEQ ID NO:9.

[0012] The second aspect of the present invention provides an application of the aforementioned monoclonal antibody in the preparation of a product for detecting ADAMTS13, wherein the application comprises using the aforementioned monoclonal antibody to detect the presence and / or content of ADAMTS13 in a biological sample.

[0013] In a preferred embodiment, the product is a kit.

[0014] The third aspect of the present invention provides an ADAMTS13 luminescent immunoassay kit, which includes reagent A, reagent B, and a luminescent substrate solution; wherein the reagent A includes the aforementioned ADAMTS13 monoclonal antibody protein-coated magnetic microparticle buffer solution; the reagent B includes an ADAMTS13 antibody solution labeled with a marker; the marker includes any one of a biological enzyme, a fluorescein, and a chemiluminescent marker; and the luminescent substrate solution includes a substrate buffer solution that can catalyze the luminescence of reagent B.

[0015] In one embodiment, the particle size of the magnetic particles coupled to ADAMTS13 antibody protein is 0.5-10 μm.

[0016] In one embodiment, the marker is a biological enzyme, including at least one of horseradish peroxidase and alkaline phosphatase; In one embodiment, the coupled bioenzyme chemiluminescent marker comprises at least one of acridinium ester, terpyridine ruthenium, isoluminol and derivatives thereof; In one embodiment, the buffer solution contained in the kit includes at least one selected from phosphate buffer, carbonate buffer, Tris buffer, and citrate buffer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 This is a flow chart of the ADAMTS13 antigen luminescent immunoassay method. DETAILED DESCRIPTION

[0018] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0019] All components of the detection kit of the present invention can be purchased from biological reagent or chemical reagent companies through commercial channels. Alkaline phosphatase (ALP) used in the present invention was purchased from BBI, UK, model: ALPI12G; SMCC was purchased from thermo fisher scientific, item number: 22360; 2-IT was purchased from thermo fisher scientific, CAS: 4781-83-3; item number: 26101; carboxyl modified microparticles were purchased from thermo fisher scientific; EDC was purchased from SIMGA, CAS: 25952-53-8, item number: E7750. ADAMTS13 antibody (R&D, USA, lot: MAB42451) Example 1: Preparation of ADAMTS-13 antigen Using the amino acid sequence of human ADAMTS13 antigen found on the NCBI official website, add a histidine tag to the N-terminus or C-terminus of the protein amino acid, optimize the codon, clone the gene into the pET30a expression vector, construct pET30a-S100A8, and sequence and verify the gene sequence. The pET30a-S100A8 expression plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and a single bacterium was selected for induction expression. It was cultured in LB medium containing 50ug / ml of kanamycin at a temperature range of 20-22℃ and 200-220rpm until OD 600 nm reached 0.6-0.8, and then 0.2mM IPTG was added for induction expression. The induction lasted for 15-18 hours, and the cells were collected by centrifugation at 8000g for 10min. The bacteria were resuspended in 20mM PB, 300mM NaCl, pH8.0 buffer, and high-pressure homogenized at 800 bar. The supernatant was centrifuged at 12000g for 30 min, and affinity purified using pre-loaded Ni Smart-6FF beads. The impurities were washed with 20mM PB300mM NaCl, 10nm imidazole, pH8.0 solution, and eluted with 20mMPB, 300mMNaCl, 250mM imidazole, pH8.0 solution. The protein purity was detected by SDS-PAGE running. The protein was dialyzed into 20mMPB, 300mM NaCl, pH8.0 buffer, and sterile filtered with a 0.22um filter. The protein concentration was determined by BCA to obtain ADAMTS13 antigen with a purity of more than 90%.

[0020] Example 2: ADAMTS-13 animal immunization Healthy Balb / c mice were selected as immunized animals, and the ADAMTS13 antigen obtained in Example 1 was used for immunization. During the first immunization, the recombinant ADAMTS-13 antigen was fully emulsified with complete Freund's adjuvant at a volume ratio of 1:1, and the mice were immunized by subcutaneous multi-point injection. The first immunization dose for each mouse was 50 μg of antigen. The first booster immunization was performed 2 weeks after the first immunization. The ADAMTS-13 antigen was emulsified with incomplete Freund's adjuvant and the mice were immunized by intraperitoneal injection, and the immunization dose was 25 μg of antigen. After that, booster immunization was performed every 2 weeks, for a total of 3-4 booster immunizations. 7 days after the last booster immunization, blood samples of the mice were collected, and the titer of the antibodies in the serum was detected by enzyme-linked immunosorbent assay (ELISA). When the antibody titer reached 1:10000 or more, it was determined that the mice had produced high-affinity ADAMTS-13 antibodies.

[0021] Example 3: Cell fusion and screening The spleen cells of immunized mice were fused with FO myeloma cells in the logarithmic growth phase under the mediation of PEG. The concentration of PEG was 50%, and the fusion conditions were gentle stirring in a 37°C water bath for 1-2 minutes, followed by the slow addition of serum-free medium to terminate the fusion reaction. The fused cell suspension was inoculated in HAT selection medium containing hypoxanthine (H), aminopterin (A) and thymine (T), and cultured in a 37°C, 5% CO2 incubator. After 1-2 weeks of culture, the unfused spleen cells and myeloma cells died because they could not grow in HAT medium, while the hybridoma cells were able to survive and proliferate. Hybridoma cell clones that could secrete anti-ADAMTS-13 antibodies were initially screened by ELISA, and then the positive clones were further subcloned by limiting dilution. After 2-3 rounds of subcloning, a monoclonal hybridoma cell line that stably secreted highly specific and high-affinity anti-ADAMTS-13 antibodies was obtained.

[0022] Example 4: Antibody purification The monoclonal hybridoma cell line obtained in Example 3 was produced by the in vivo ascites preparation method or the in vitro cell culture method. The in vivo ascites preparation method is to inoculate the hybridoma cells into the peritoneal cavity of pretreated Balb / c mice, with 1×10 6The ascites is collected about 7-10 days later, and the antibody content in the ascites can reach 5-10 mg / ml. The in vitro cell culture method is to culture the hybridoma cells in a large-scale suspension in a bioreactor, and to increase the yield of the antibody by optimizing the culture conditions (such as adjusting the culture medium composition, controlling the dissolved oxygen and stirring speed, etc.). The antibody is purified from the ascites or cell culture supernatant, and the anti-ADAMTS-13 antibody is specifically bound to the Protein A or Protein G affinity column by affinity chromatography, and then eluted with elution buffer to obtain an anti-ADAMTS-13 monoclonal antibody with a purity greater than 98%. The purity and specificity are identified by SDS-PAGE electrophoresis and Western Blot, and the affinity of the anti-ADAMTS-13 monoclonal antibody to the ADAMTS13 antigen is tested by ELISA technology to ensure that the prepared antibody has good detection performance. The sequence information of the ADAMTS13 antibody is shown in Table 1.

[0023] Table 1 Sequence information of ADAMTS13 antibody

[0024]

[0025] Example 5: Magnetic microparticle labeling of ADAMTS13 antibody Take 20 mg of carboxyl-modified microparticle solution, and let the magnetic microparticles with superparamagnetism, uniform particle size and carboxyl (C00H-) active groups on the surface settle under the action of a magnetic field (magnetic separation) for 10 minutes, remove the supernatant, and wash the settled magnetic microparticles three times with an activation buffer with a molar concentration of 0.05 M (2-(N-morpholine)ethanesulfonic acid) MES, pH 6.0 buffer, each time using 2 ml.

[0026] The washed magnetic particles were fully suspended with 1.0 ml of activation buffer (2-(N-morpholine)ethanesulfonic acid) MES, pH 6.0, with a molar concentration of 0.05 M, and then the activator 1-ethy1-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was added. The suspension was reacted at room temperature for 30 minutes. The reaction molar concentration of EDC was 7.5 mM.

[0027] Example 6: Alkaline phosphatase labeled ADAMTS13 antibody

[0028] Take 1.0 mg of commercial ADAMTS13 antibody (R&D, USA, lot: MAB42451), add it to the activated magnetic particle solution with a magnetic bead content of 20 mg at a ratio of 1:20, shake it gently to mix, and react it at 4°C suspension conditions for 6.5 hours to make the ADAMTS13 antibody covalently coupled to the surface of the magnetic particles to prepare reagent A.

[0029] Example 7: ADAMTS13 magnetic microparticle separation chemiluminescent immunoassay (1) Immune reaction: 50 μl of the calibrator series (with concentrations of 0 ng / ml, 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, and 20 ng / ml) or the quality control sample of Example 1 and 50 μl of reagent A of Example 5 and 50 μl of reagent B of Example 6 were added to the reaction tubes in sequence, and mixed and incubated at 37°C for 10 min; (2) Magnetic separation: allow the magnetic particles to settle in a magnetic field, remove the supernatant, add 200-500 μl of cleaning solution, remove the magnetic field, and then allow the magnetic particles to settle in a magnetic field again, and remove the supernatant; repeat this process 2-4 times to remove unbound antibodies and impurities; (3) Reading: Add 200 μl of luminescent substrate solution, and use a chemiluminescence detector to measure the relative luminescence intensity (RLU) after alkaline phosphatase catalyzes the substrate to emit light; (4) A four-parameter equation was used to fit the detected values ​​to obtain the ADAMTS13 concentration-luminescence value standard curve.

[0030] (5) 50 μl of the sample to be tested, 50 μl of reagent A in Example 1, and 50 μl of reagent B in Example 2 were added to the reaction tube in sequence, and mixed and incubated at 37°C for 15 min; (6) Magnetic separation: allow the magnetic particles to settle in a magnetic field, remove the supernatant, add 200-500 μl of cleaning solution, remove the magnetic field, and then allow the magnetic particles to settle in a magnetic field again, and remove the supernatant; repeat this process 2-4 times to remove unbound antibodies and impurities; (7) Reading: Add 200 μl of luminescent substrate solution, and use a chemiluminescence detector to measure the relative luminescence intensity (RLU) after ALP catalyzes the substrate to emit light; (8) The luminescence intensity of the sample to be tested is compared with the standard curve in step (4), and the content of ADAMTS13 in the sample to be tested can be calculated using a four-parameter equation.

[0031] Example 8: Kit Precision Performance Test The test was repeated 10 times using samples of high and low concentration levels and the kit described in Example 7, and the mean (range) and coefficient of variation (CV) of the test results were calculated according to formulas 1 and 2. The test results are shown in Table 2.

[0032]

[0033] Where Xi represents the measurement result of the sample to be tested, represents the mean of the sample to be tested, n represents the number of measurements, which is 10.

[0034] Table 2 Kit precision test results

[0035] The results in Table 2 show that the mean values ​​of 10 repeated tests using the kit of the present invention are relatively close, and the coefficient of variation of the kit is less than 10%, indicating that the kit of the present invention has high precision.

[0036] Example 9: Kit linear range test The highest concentration point calibrator of the kit described in Example 7 and the dilution of the calibrator of the project were diluted to a concentration close to the analytical sensitivity of the project to form a sample of the ADAMTS13 antigen solution concentration point in the range of 0-50 ng / mL. After the kit was calibrated, the gradient samples were tested, and each dilution concentration was tested 3 times, and the mean (y) of the measurement results was calculated respectively. The linear regression equation was calculated with the dilution concentration (x) as the independent variable and the mean (y) of the measurement result as the dependent variable. The correlation coefficient (r) of the linear regression was calculated according to formula (3).

[0037]

[0038] Table 3 Dose-response curve linearity determination

[0039] The results in Table 2 show that the r value of the linear reaction curve of the kit prepared by the present invention is >0.9900 in the range of 0-50 ng / ml.

[0040] Example 10: Kit Clinical Sample Testing The kit described in Example 7 was used in combination with a fully automatic chemiluminometer (Chongqing Cosmay, lot: KSM22H50706) to detect 7 plasma samples from TTP patients and 7 plasma samples from normal people. The test results are shown in Table 4. By comparison, it can be seen that the kit prepared by the present invention can effectively distinguish normal human plasma samples from TTP patient plasma samples. The luminescence values ​​of the two groups of samples were statistically analyzed by T test, and the results showed significant differences (P<0.05).

[0041] Table 4 Test results of clinical samples of the kit

[0042] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An anti-ADAMTS13 monoclonal antibody, characterized in that: The monoclonal antibody comprises a light chain sequence and a heavy chain sequence, wherein the light chain sequence comprises CDR1 as shown in SEQ ID NO.3, CDR2 as shown in SEQ ID NO:4, and CDR3 as shown in SEQ ID NO:5, and the heavy chain sequence comprises CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:7, and CDR3 as shown in SEQ ID NO:

8.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises a light chain variable region amino acid sequence as shown in SEQ ID NO.1, and a heavy chain variable region amino acid sequence as shown in SEQ ID NO.

2.

3. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody further comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian antibody; optionally, the heavy chain constant region and the light chain constant region are derived from at least one of mouse, human, primate, bovine, horse, dairy cow, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink antibodies or mutants thereof.

4. The monoclonal antibody according to claim 3, characterized in that The heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ or λ; optionally, the heavy chain constant region and the light chain constant region are both derived from murine antibodies or mutants thereof; optionally, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

5. The monoclonal antibody according to claim 3 or 4, characterized in that The nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:10, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:

9.

6. Use of the monoclonal antibody according to any one of claims 1 to 5 in preparing a product for detecting ADAMTS13, characterized in that: The application includes using the monoclonal antibody as described in any one of claims 1 to 5 to detect the presence and / or content of ADAMTS13 in a biological sample.

7. The use according to claim 6, characterized in that The product is a test kit.

8. An ADAMTS13 luminescent immunoassay kit, characterized in that: The kit comprises reagent A, reagent B, and a luminescent substrate solution; wherein the reagent A comprises a magnetic microparticle buffer solution coated with the ADAMTS13 monoclonal antibody protein as described in any one of claims 1 to 5; the reagent B comprises an ADAMTS13 antibody solution labeled with a marker; the marker comprises any one of a biological enzyme, a fluorescein, and a chemiluminescent marker; and the luminescent substrate solution comprises a substrate buffer solution that can catalyze the luminescence of the reagent B.

9. The kit according to claim 8, characterized in that: The particle size of the magnetic particles coupled to the ADAMTS13 antibody protein is 0.5-10 μm; The marker is a biological enzyme, including at least one of horseradish peroxidase and alkaline phosphatase; The coupled bioenzyme chemiluminescent marker includes at least one of acridinium ester, terpyridine ruthenium, isoluminol and derivatives thereof; The buffer solution contained in the kit includes at least one selected from phosphate buffer, carbonate buffer, Tris buffer and citrate buffer.

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