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Monoclonal antibodies resistant to PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and application of monoclonal antibodies

A technology of monoclonal antibody and deposit number, which is applied in the field of monoclonal antibody and can solve problems such as the lack of effective means

Active Publication Date: 2016-07-13
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is still a lack of effective means for how to prevent and control tumor metastasis. Preventing and inhibiting tumor metastasis will be the focus, hot spot and difficulty in tumor prevention and treatment for a long time

Method used

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  • Monoclonal antibodies resistant to PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and application of monoclonal antibodies
  • Monoclonal antibodies resistant to PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and application of monoclonal antibodies
  • Monoclonal antibodies resistant to PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and application of monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Immunogen preparation—a 22-amino acid polypeptide fragment (DTETTGLEGGVAMPGAEDDVVC) of the PLAG region of artificially synthesized human PDPN (see mass spectrogram figure 1 ) was coupled with keyhole limpet hemocyanin (KLH) to form DTETTGLEGGVAMPGAEDDVVC-KLH as an immunogen, and Balb / c mice were immunized. (Completed by Shanghai Ziyu Biotechnology Co., Ltd.)

Embodiment 2

[0041] Embodiment 2: the preparation of the monoclonal antibody of the PLAG region of specific anti-PDPN

[0042] We prepared the monoclonal antibody of the present invention using conventional immunological methods and hybridoma technology (Köhler and Milstein, Nature, 215:495-497, 1975).

[0043] First, 8-week-old female Balb / c mice (Experimental Animal Center, Shanghai Academy of Sciences) were immunized with artificially synthesized conjugated 22-peptide-KLH (DTETTGLEGGVAMPGAEDDVVC-KLH) 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.

[0044] After the serum antibody titer of the immunized mice reached a high enough level, the animals were sacrificed and spleen cells were isolated. Using standard monoclonal antibody cell fusion technology (Kǒhler and Milstein, Nature, 215: 495-4...

Embodiment 3

[0047] Example 3: Biochemical properties of monoclonal antibodies of the present invention

[0048] (1) It was confirmed by the double-diffusion method that the monoclonal antibodies SZ-163 and SZ-168 of the present invention belong to the IgG1 subclass.

[0049] (2) Inoculate the clones producing monoclonal antibodies SZ-163 and SZ-168 in the peritoneal cavity of Balb / c mice to produce ascites, and use protein G-Sepharose-4B ( Pharmacia Product) column purification, every 1mL of ascites can be purified to obtain 1.0mg of IgG.

[0050] (3) Determination of the specificity of SZ-163 and SZ-168 by ELISA experiment: the artificially synthesized PDPN 22 peptide (1 μg / ml) was coated overnight at 4°C, washed with PBS-0.05%-Tween 20, and washed with 2% BSA-PBS blocked overnight. Dilute SZ-163 and SZ-168 from 1 μg / ml to 6 concentrations (1 μg / ml, 500ng / ml, 250ng / ml, 125ng / ml, 62.5ng / ml, 31.25ng / ml), 37°C React for 1.5 hours. After washing, horseradish peroxidase-labeled goat anti...

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Abstract

The invention discloses monoclonal antibodies resistant to a PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and an application of the monoclonal antibodies. The monoclonal antibodies are secreted by hybridoma cell lines with preservation numbers of CCTCC NO: C201613 and CCTCC NO: C2015185 respectively, are named as SZ-163 and SZ-168 and are both IgG1 subclass antibodies; the monoclonal antibodies are specifically bound with reductive PDPN, are bound with an NCI-H226 cell Lysis solution to generate a binding zone when the molecular weight is 36 kDa, and are bound with a recombinant human PDPN-Fc to generate a binding zone when the molecular weight is 67 kDa. One anti-PDPN monoclonal antibody can block platelet activating aggregation induced through binding of tumor cells and platelets, and the other anti-PDPN monoclonal antibody can be bound with the first functional inhibiting antibody at different epitopes of PDPN. The antibodies can be used for preparing an anti-tumor-metastasis drug or establishing a double-antibody sandwich method for measuring the content of soluble PDPN in human plasma.

Description

technical field [0001] The present invention relates to monoclonal antibodies SZ-163 and SZ-168 against human podoplanin (Podoplanin, PDPN) stimulating platelet aggregation domain (PLateletAGgregation-stimulating, PLAG) functional region and preparation methods thereof; the present invention further relates to the prepared function The application of anti-antibodies in the preparation of drugs for inhibiting tumor growth and metastasis; the invention relates to the application of monoclonal antibodies in the establishment of double-antibody sandwich ELISA to detect human plasma soluble PDPN. Background technique [0002] Due to factors such as environment, diet, living habits, work pressure, and genetics, the incidence and mortality of tumors have remained high. Tumors have become common and frequently occurring diseases that seriously endanger human health. Tumor metastasis is a major cause of death in cancer patients. Although the mechanism of tumor metastasis has been pa...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/574A61K39/395A61P35/04
CPCA61K2039/505C07K16/18C07K2317/73
Inventor 赵益明夏利军赵星鹏傅建新阮长耿沈飞
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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