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Inhibitory monoclonal antibody resistant to lyase of human von Willebrand factor (VWF) and application of inhibitory monoclonal antibody

A monoclonal antibody, hemophilia factor technology, applied in the field of immunology to achieve the effect of increasing targeting and specificity

Active Publication Date: 2016-10-12
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The specific function of each structure of ADAMTS13 has not yet been fully elucidated. Therefore, the development of monoclonal antibodies against ADAMTS13 will provide a powerful research tool for the study of ADAMTS13 function, mechanism of thrombosis, and new treatment methods for TTP and type 2A VWD.

Method used

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  • Inhibitory monoclonal antibody resistant to lyase of human von Willebrand factor (VWF) and application of inhibitory monoclonal antibody
  • Inhibitory monoclonal antibody resistant to lyase of human von Willebrand factor (VWF) and application of inhibitory monoclonal antibody
  • Inhibitory monoclonal antibody resistant to lyase of human von Willebrand factor (VWF) and application of inhibitory monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Preparation of immunogen—recombinant human von Willebrand factor lyase truncated protein ADAMTS13-T7

[0031] (1.1) Transformation amplification and quantification of eukaryotic expression vectors

[0032] The pSecTag-ADAMTS13-T7 plasmid was transformed into E. coli Escherchia coli DH5α competent, and incubated overnight at 37°C after plating. Pick positive clones, amplify and culture overnight on a bacterial shaker at 37°C, collect bacteria by centrifugation at 4°C, and extract plasmids with a plasmid extraction kit (operate according to the kit instructions). The plasmid was identified by Hind III and Xho I double enzyme digestion.

[0033] (1.2) Transfection of HeLa cells with pSecTag-ADAMTS13-T7 plasmid

[0034] HeLa cell culture: HeLa cells were cultured in DMEM (Dulbecco's Modified Eagles Medium) containing 10% calf serum. 24 hours before transfection, the cells were seeded in 24 cell culture well plates (1.0 × 10 5 / mL), to 60-80% fusion.

[0035] ...

Embodiment 2

[0056] Example 2 Preparation of Monoclonal Antibody Specific Anti-Von Willebrand Factor Lyase

[0057] The monoclonal antibody of the present invention can be prepared using conventional immunological methods and hybridoma technology (Köhler and Milstein, Nature, 215:495-497, 1975).

[0058] First, 8-week-old female Balb / c mice (Experimental Animal Center, Shanghai Academy of Sciences) were immunized with purified ADAMTS13-T7 protein for three times with an interval of four weeks. The first two times were multi-point subcutaneous injection on the back plus intraperitoneal injection, and the third time was injection through the mouse tail vein plus intraperitoneal injection.

[0059] After the serum antibody titer of the immunized mice reached a high enough level, the animals were sacrificed and spleen cells were isolated. Using the standard monoclonal antibody cell fusion technology, the spleen cells of the immunized Balb / c mice were fused with the mouse SP2 / 0 myeloma cells (...

Embodiment 3

[0062] Embodiment 3 detects the chemical property of monoclonal antibody

[0063] (1) It was confirmed by the double-diffusion method that the monoclonal antibody SZ-169 of the present invention belongs to the IgG1 subclass.

[0064] (2) The monoclonal antibody SZ-169 was inoculated in the peritoneal cavity of Balb / c mice to produce ascites, which was purified by BeaverBeadsTM ProteinA / G Matrix antibody purification magnetic beads, dialyzed in PBS, and the IgG content was measured with a UV spectrophotometer. Ascites can be purified to obtain IgG 3-6mg.

[0065] (3) Determination of the specificity of SZ-169 by ELISA experiment: the titer of the purified antibody was detected by ELISA method. Purified ADAMTS13-T7 protein and ADAMTS13 full-length protein were prepared in carbonate buffer to 1 μg / mL, 100 μl / well plated, overnight at 4°C; washed twice with 0.05% Tween-PBS, blocked overnight with 2% BSA-PBST , after washing as above, add 100 μl of purified antibody (1:100, 1:100...

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Abstract

The invention discloses an inhibitory monoclonal antibody resistant to lyase of a human von Willebrand factor (VWF) and application of the inhibitory monoclonal antibody. The monoclonal antibody belongs to an IgG1 subclass antibody and is produced from a hybridoma cell strain SZ-169; the preservation number of the hybridoma cell strain SZ-169 is CCTCC NO: C2016134. Compared with the prior art, the inhibitory monoclonal antibody has the following advantages: (1) the monoclonal antibody can be specifically combined with human overall-length ADAMTS13 and truncated ADAMTS13-T7 proteins, and the bonding capacity with the truncated ADAMTS13-T7 proteins is obviously higher than that with the overall-length ADAMTS13 proteins; (2) under a denaturation condition, the monoclonal antibody can obviously prevent ADAMTS13 from hydrolyzing VWF , and the suppression effect is enhanced with the increase of the concentration of the monoclonal antibody; (3) the monoclonal antibody disclosed by the invention can be effectively applied to medicine preparation, so that the targeting property and the specificity of a medicine are improved.

Description

technical field [0001] The invention belongs to the field of immunology and relates to a monoclonal antibody, in particular to an inhibitory monoclonal antibody against human von Willebrand factor lyase and application thereof. Background technique [0002] Von Willebrand factor cleavage protease (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13, ADAMTS13) is a metalloprotease purified from plasma, belonging to the metalloproteinase superfamily (a disintegrin and metalloproteinase with thrombospondin motifs, ADAMTSs) . ADAMTS13 inhibits platelet aggregation and microvascular thrombosis by cleaving the Tyr1605-Met1606 peptide bond of VWF and reducing the molecular weight of its substrate von Willebrand factor (VWF). Studies have shown that congenital or acquired defects of ADAMTS13 lead to disseminated microvascular thrombus, which is closely related to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Thrombotic thrombocytopenic purpu...

Claims

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Application Information

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IPC IPC(8): C07K16/40A61K39/395A61P7/04
CPCA61K39/00C07K16/40
Inventor 马珍妮凌婧沈飞冯宇锋苏健阮长耿
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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