Antibodies for adamts-13 detection and use thereof

Abstract

The present application relates to the field of antibodies, in particular, the present application relates to a kind of antibody for ADAMTS-13 detection and its application.The ADAMTS13 monoclonal antibody provided by the present application, the light chain sequence of the monoclonal antibody includes CDR1 shown as SEQ ID NO.3, CDR2 shown as SEQ ID NO:4, CDR3 shown as SEQ ID NO:5;Heavy chain sequence includes CDR1 shown as SEQ ID NO:6, CDR2 shown as SEQ ID NO:7 and CDR3 shown as SEQ ID NO:8.In addition, the present application also provides detection kit comprising the monoclonal antibody, which can effectively detect the presence and / or content of ADAMTS13 in biological sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular, the present application relates to an antibody for ADAMTS-13 detection and application thereof. BACKGROUND

[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 cleaving vWF multimers, thereby delaying thrombus formation.

[0003] Accurate measurement of ADAMTS13 antigen level is crucial for assessing the thrombosis risk of individuals. The ratio of vWF / ADAMTS13 can predict the thrombotic vascular risk of patients and assess the recovery of patients after treatment and exclude the risk of thrombosis in time. At the same time, the detection of ADAMTS13 antigen level together with the detection of ADAMTS13 autoantibody and ADAMTS13 enzyme activity can assist the diagnosis of thrombocytopenic purpura (TPP).

[0004] The more mature ADAMTS13 antigen detection method is ELISA method, which has the problems of long detection time, complex operation, poor repeatability and specificity. Therefore, a detection method with high sensitivity, simple operation, high automation degree and low cost is still needed. SUMMARY

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

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

[0007] In an embodiment, 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.

[0008] In an embodiment, 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 which 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 a murine, human, primate, bovine, equine, dairy cattle, porcine, ovine, caprine, canine, feline, lagomorph, camelid, donkey, cervid, mink antibody or a mutant thereof.

[0009] In a preferred embodiment, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region comprises a light chain constant region selected from kappa or lambda; optionally, the heavy chain constant region and the light chain constant region are derived from a murine antibody or a mutant 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 an embodiment, the antibody belongs to the IgG1 subclass, and the heavy chain constant region and the light chain constant region thereof have the amino acid sequence characteristics of a native human IgG1 antibody.

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

[0012] In a second aspect of the present application, there is provided a use of the aforementioned monoclonal antibody in the preparation of a product for detecting ADAMTS13, which use comprises using the aforementioned monoclonal antibody to detect the presence and / or amount of ADAMTS13 in a biological sample.

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

[0014] In a third aspect of the present application, there is provided an ADAMTS13 luminescent immunoassay kit, comprising reagent A, reagent B, and a luminescent substrate solution; wherein the reagent A comprises a magnetic particle buffer solution coated with the aforementioned ADAMTS13 monoclonal antibody protein; the reagent B comprises a solution of an ADAMTS13 antibody labeled with a label; the label comprises any one of a biological enzyme, a fluorescein, and a chemiluminescent label; and the luminescent substrate solution comprises a substrate buffer solution capable of catalyzing luminescence of the reagent B.

[0015] In an embodiment, the particle size of the ADAMTS13 antibody protein-magnetic particle conjugate is 0.5-10 μm.

[0016] In an embodiment, the label is a biological enzyme, and comprises at least one of horseradish peroxidase and alkaline phosphatase.

[0017] In an embodiment, the biological enzyme chemiluminescent label comprises at least one of acridinium ester, trispyridine ruthenium, and isoluminol and derivatives thereof.

[0018] In an embodiment, the buffer solution contained in the kit comprises at least one selected from the group consisting of a phosphate buffer, a carbonate buffer, a tris buffer, and a citrate buffer. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, illustrate embodiments of the present application, and are used to explain the present application, but do not limit the present application. In the drawings:

[0020] Figure 1 Flow chart of the ADAMTS13 antigen luminescent immunoassay method. DETAILED DESCRIPTION

[0021] The solutions of the present disclosure will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only for the purpose of illustrating the present disclosure, and should not be considered as limiting the scope of the present disclosure. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained commercially.

[0022] All components in the detection kit of the present application can be purchased from biological reagent or chemical reagent companies through commercial channels. The alkaline phosphatase (ALP) used in the present application is purchased from BBI Company in the United Kingdom, model number: ALPI12G; SMCC is purchased from thermo fisher scientific company, catalog number: 22360; 2-IT is purchased from thermo fisher scientific company, CAS: 4781-83-3; catalog number: 26101; carboxyl modified microparticles are purchased from thermo fisher scientific company; EDC is purchased from SIMGA company, CAS: 25952-53-8, catalog number: E7750. ADAMTS13 antibody (R&D, USA, lot: MAB42451)

[0023] Example 1: Preparation of ADAMTS-13 antigen

[0024] The amino acid sequence of human ADAMTS13 antigen searched on the NCBI website is added with a histidine tag at the N or C terminal of the protein amino acid, and after optimization of the codon, the gene is cloned into the pET30a expression vector to construct pET30a-S100A8, and the gene sequence is verified by sequencing. The pET30a-S100A8 expression plasmid is transformed into E. coli BL21 (DE3) competent cells, single bacteria are picked for induction and expression, cultured in LB medium containing 50 ug / m1 kanamycin, and the culture temperature is 20-22℃, 200-220 rpm. When the OD 600 nm is 0.6-0.8, 0.2mM IPTG is added for induction and expression, and the induction is carried out for 1518 hours, 8000g, centrifugation for 10min, and the bacterial body is collected. The bacterial body is resuspended with 20mM PB, 300mM NaC1, pH8.0 buffer, and homogenized under high pressure of 800bar, then centrifuged at 12000g for 30min, and the supernatant is collected. The pre-packed column Ni Smart-6FFbeads is used for affinity purification, 20mM PB300mM NaC1, 10nm imidazole, pH8.0 solution is used for washing, 20mM PB, 300mM NaCl, 250mM imidazole, pH8.0 solution is used for elution, SDS-PAGE is used for detecting protein purity, the protein is dialyzed into 20mM PB, 300mM NaC1, pH8.0 buffer, 0.22um filter is used for sterile filtration, BCA is used for determining protein concentration, and ADAMTS13 antigen with a purity of more than 90% is obtained.

[0025] Example 2: ADAMTS-13 animal immunization

[0026] Healthy Balb / c mice were selected as immunized animals, and the ADAMTS13 antigen obtained in Example 1 was used for immunization. For the first immunization, the recombinant ADAMTS-13 antigen was emulsified with complete Freund's adjuvant at a volume ratio of 1:1, and the mice were immunized by subcutaneous multiple point injection. The first immunization dose of each mouse was 50 μg of antigen. Two weeks after the first immunization, the first booster immunization was performed, and the mice were immunized intraperitoneally with ADAMTS-13 antigen emulsified with incomplete Freund's adjuvant, with an immunization dose of 25 μg of antigen. Then, booster immunization was performed every 2 weeks, for a total of 3-4 times. Seven days after the last booster immunization, the blood samples of the mice were collected, and the antibody titer in the serum was detected by enzyme-linked immunosorbent assay (ELISA). When the antibody titer reached more than 1:10,000, it was determined that the mouse had produced high-affinity ADAMTS-13 antibodies.

[0027] Example 3: Cell fusion and screening

[0028] The spleen cells of the 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 slow addition of serum-free medium to terminate the fusion reaction. The cell suspension after fusion was inoculated in HAT selection medium containing hypoxanthine (H), aminopterin (A), and thymidine (T), and cultured in a 37°C, 5% CO2 incubator. After 1-2 weeks of culture, un-fused spleen cells and myeloma cells died because they could not grow in HAT medium, while hybridoma cells could survive and proliferate. Hybridoma cell clones that could secrete anti-ADAMTS-13 antibodies were preliminarily screened by ELISA, and then the positive clones were further subcloned and screened by limiting dilution method. After 2-3 rounds of subcloning, a single monoclonal hybridoma cell strain stably secreting high-specificity and high-affinity anti-ADAMTS-13 antibodies was obtained.

[0029] Example 4: Antibody purification

[0030] The monoclonal hybridoma cell strain obtained in Example 3 was produced by in vivo ascites preparation method or in vitro cell culture method. The in vivo ascites preparation method was to inoculate the hybridoma cells into the peritoneal cavity of a pretreated Balb / c mouse, with 1×10 6The hybridoma cells are cultured in vitro for about 7-10 days to collect the ascites, and the antibody content in the ascites can reach 5-10 mg / ml. In vitro cell culture method is to culture hybridoma cells in a bioreactor for large-scale suspension culture, and to improve the yield of antibodies by optimizing the culture conditions such as adjusting the medium composition, controlling the dissolved oxygen and stirring speed, etc. The antibody is purified from ascites or cell culture supernatant, and affinity chromatography is used to specifically bind the anti-ADAMTS-13 antibody to the Protein A or Protein G affinity column, and then eluted with elution buffer to obtain anti-ADAMTS-13 monoclonal antibody with a purity of more than 98%. The purity and specificity of the antibody are identified by SDS-PAGE electrophoresis and Western Blot, and the affinity of the anti-ADAMTS-13 monoclonal antibody to 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.

[0031] Table 1 Sequence information of ADAMTS13 antibody

[0032]

[0033]

[0034] Example 5: Magnetic microparticle-labeled ADAMTS13 antibody

[0035] Take 20 mg of carboxyl-modified microparticle solution, and the magnetic microparticles with superparamagnetic, uniform particle size, and carboxyl (COOH-) active groups on the surface are settled (magnetic separation) under the action of a magnetic field for 10 minutes. The supernatant is removed, and the settled magnetic microparticles are washed with an activation buffer solution of 0.05 M 2- (N-morpholine) ethanesulfonic acid) MES, pH 6.0 buffer solution for 3 times, each time with an amount of 2 ml.

[0036] After washing the magnetic microparticles, 1.0 ml of the activation buffer solution of 0.05 M 2- (N-morpholine) ethanesulfonic acid) MES, pH 6.0 buffer solution is fully suspended, and then an activation agent 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) is added. The suspension is reacted at room temperature for 30 minutes, and the reaction molar concentration of EDC is 7.5 mM.

[0037] Example 6: Alkaline phosphatase-labeled ADAMTS13 antibody

[0038] Take 1.0 mg of commercial ADAMTS13 antibody (R&D, USA, lot: MAB42451), add to the activated magnetic microsphere solution at a ratio of 1:20, mix gently, and react for 6.5 hours at 4°C under suspension conditions to allow the ADAMTS13 antibody to be covalently coupled to the surface of the magnetic microspheres, thereby preparing reagent A.

[0039] Example 7: ADAMTS13 magnetic microsphere separation chemiluminescence immunoassay method

[0040] (1) Immune reaction: 50 μl of the calibration sample series of Example 1 (concentrations of 0 ng / ml, 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, and 20 ng / ml, respectively) or the quality control sample were added to the reaction tube in turn with 50 μl of reagent A of Example 5 and 50 μl of reagent B of Example 6, and mixed and incubated at 37°C for 10 min;

[0041] (2) Magnetic separation: the magnetic microspheres were allowed to settle in a magnetic field, the supernatant was removed, 200-500 μl of washing solution was added, the magnetic field was removed, and then the magnetic microspheres were again allowed to settle in a magnetic field, and the supernatant was removed; this was repeated 2-4 times to remove unbound antibodies and impurities;

[0042] (3) Reading: 200 μl of luminescent substrate solution was added, and the relative luminescence intensity (RLU) was measured using a chemiluminescence detector after the substrate was luminescent under the catalysis of alkaline phosphatase;

[0043] (4) The ADAMTS13 concentration-luminescence value standard curve was obtained by fitting the detected values using a four-parameter equation.

[0044] (5) 50 μl of the sample to be tested was added to the reaction tube in turn with 50 μl of reagent A of Example 1 and 50 μl of reagent B of Example 2, and mixed and incubated at 37°C for 15 min;

[0045] (6) Magnetic separation: the magnetic microspheres were allowed to settle in a magnetic field, the supernatant was removed, 200-500 μl of washing solution was added, the magnetic field was removed, and then the magnetic microspheres were again allowed to settle in a magnetic field, and the supernatant was removed; this was repeated 2-4 times to remove unbound antibodies and impurities;

[0046] (7) Reading: 200 μl of luminescent substrate solution was added, and the relative luminescence intensity (RLU) was measured using a chemiluminescence detector after the substrate was luminescent under the catalysis of alkaline phosphatase;

[0047] (8) The luminescence intensity of the sample to be tested was compared with the standard curve of step (4), and the content of ADAMTS13 in the sample to be tested was calculated using a four-parameter equation.

[0048] Example 8: Kit precision performance test

[0049] The sample with high and low concentration levels and the kit described in Example 7 were tested 10 times, and the mean (region) and coefficient of variation (CV) of the determination results were calculated according to Formulas 1 and 2. The determination results are shown in Table 2.

[0050]

[0051] In the formula, Xi represents the determination result of the sample to be tested, represents the mean of the sample to be tested, and n represents the number of determinations, which is 10.

[0052] Table 2 Kit precision test results

[0053]

[0054] As shown by the results in Table 2, the mean of 10 repeated tests using the kit of the present application is close, and the coefficient of variation of the kit is less than 10%, indicating that the kit of the present application has high precision.

[0055] Example 9: Kit linear range test

[0056] The highest concentration point calibrator of the kit described in Example 7 and the dilution solution of the project calibrator were diluted to close to the analysis sensitivity of the project to form ADAMTS13 antigen solution concentration point samples in the range of 0-50 ng / mL. After the kit was calibrated, the gradient samples were tested, each dilution concentration was tested 3 times, and the mean (y) of the determination results was calculated. The linear regression equation was calculated with the dilution concentration (x) as the independent variable and the mean (y) of the determination results as the dependent variable. The correlation coefficient (r) of linear regression was calculated according to Formula (3).

[0057]

[0058] Table 3 Dose-response curve linear determination

[0059]

[0060] As shown by the results in Table 2, the mean of 10 repeated tests using the kit of the present application is close, and the coefficient of variation of the kit is less than 10%, indicating that the kit of the present application has high precision.

[0061] Example 10: Kit clinical sample test

[0062] The kit of Example 7 was used in combination with a full-automatic chemiluminescence instrument (Chongqing Kosmoe, lot: KSM22H50706) to detect 7 plasma samples of TTP patients and 7 plasma samples of normal persons. The detection results are shown in Table 4. It can be seen by comparison that the kit prepared in the application can effectively distinguish the plasma samples of normal persons and the plasma samples of TTP patients. The luminescence values of the two groups of samples were statistically analyzed by T test, and the results had a significant difference (P < 0.05).

[0063] Table 4 Kit clinical sample detection results

[0064]

[0065] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application belong to the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. An anti-ADAMTS 13 monoclonal antibody, characterized in that, The monoclonal antibody comprises a light chain sequence and a heavy chain sequence, wherein the light chain sequence comprises a CDR1 as shown in SEQ ID NO. 3, a CDR2 as shown in SEQ ID NO: 4, and a CDR3 as shown in SEQ ID NO: 5, and 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.

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 or 2, 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.

4. Use of a monoclonal antibody according to any one of claims 1 to 3 for the manufacture of a product for the detection of ADAMTS 13, characterized in that The application comprises using the monoclonal antibody according to any one of claims 1-3 to detect the content or activity of ADAMTS13 in a biological sample in vitro.

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