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Monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of LMP2A extracellular region and use thereof

A monoclonal antibody and fusion protein technology, applied in the field of biomedicine and immune targeted diagnosis and treatment, can solve the problems of reduced sensitivity to radiotherapy and chemotherapy, easy recurrence and metastasis, and unsatisfactory survival rate, and achieve high sensitivity and specificity. Effect

Inactive Publication Date: 2015-12-16
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, no effective treatment plan has been found for nasopharyngeal carcinoma, which is prone to metastasis. Although the condition of most patients has improved after comprehensive treatment such as radiotherapy and chemotherapy, the survival rate after 5 years is still very unsatisfactory, and Very prone to recurrence and metastasis, especially after recurrence, the sensitivity of radiotherapy and chemotherapy is greatly reduced

Method used

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  • Monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of LMP2A extracellular region and use thereof
  • Monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of LMP2A extracellular region and use thereof
  • Monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of LMP2A extracellular region and use thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Preparation and identification of anti-LMP2A monoclonal antibody:

[0024] 1. Antigen Immunization

[0025] Antigen emulsification: Freund's complete adjuvant was pre-heated slightly, sucked 200 μL into a 1 mL sterilized EP tube with a syringe, dripped 200 μL of purified LMP2A extracellular region epitope tandem fusion protein, and shaken while dripping until the emulsification was complete, inoculated into mice . Mice were immunized with complete Freund's adjuvant for the first time, and Freund's incomplete adjuvant for subsequent immunizations. Wherein the amount of adjuvant and protein is 1:1.

[0026] Initial immunization: After mixing 200 μL MP2A antigen (100 μg) with an equal amount of Freund’s complete adjuvant, 200 μL / mouse was subcutaneously injected into BALB / c mice at multiple points on the back of the neck and marked;

[0027] Booster immunization: mix the same dose of antigen with Freund's incomplete adjuvant, immunize every other week, the immunization ...

Embodiment 2

[0085] Analysis of immunological characteristics of anti-LMP2A monoclonal antibody:

[0086] Identification of Monoclonal Antibodies by Westernblotting

[0087] 1) Culture cells B95-8, SUNE, CNE, U937, NIH3T3 with 1640 medium containing 1% PS and 10% FBS. Cultured at 37°C, 5% CO 2 in the incubator.

[0088] 2) Observe the growth status of the cells every day, and change the medium regularly. The cells were immediately subcultured when they were congested, and some of them were frozen in liquid nitrogen.

[0089] 3) After the cells are congested, digest the cells with trypsin, collect the digested cells into a 15 mL centrifuge tube, and wash them 1-2 times with high-pressure sterilized PBS.

[0090] 4) Centrifuge at 1000 rpm for 5 min. repeat three times.

[0091] 5) The cell pellet was resuspended in 20 μL PBS, mixed with an equal volume of 2×loadingbuffer and heated, then loaded with a constant voltage of 80V for about 1 hour.

[0092] 6) Transfer to PVDF membrane, cur...

Embodiment 3

[0105] Detection of LMP2A expression in nasopharyngeal carcinoma tumor tissue by anti-LMP2A monoclonal antibody:

[0106] Immunohistochemical detection

[0107] 1) 33 cases of nasopharyngeal carcinoma were selected from the Second Affiliated Hospital of Nanjing Medical University and Jiangsu Cancer Hospital. The slices were 4 μm thick, baked in an oven at 60°C for 1 hour before dewaxing, and then dewaxed and hydrated.

[0108] 2) Wash with PBS 3 times, each time for 5min, H 2 o 2 Effect at room temperature for 5 to 10 minutes.

[0109] 3) After washing with PBS for 3 times, heat in a microwave oven at high temperature to restore the antigen.

[0110] 4) Rinse gently with PBS, soak in PBS for 5 minutes, repeat three times.

[0111] 5) Add 5% calf serum dropwise to block, block at room temperature for 30 minutes, shake off the serum, add the prepared monoclonal antibody 5C12-C4-A6 dropwise, dilute the antibody to a final concentration of 0.25 μg / mL, and use SP2 / 0 cell ascite...

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Abstract

The invention discloses a monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of a LMP2A extracellular region and a use thereof. The monoclonal antibody is produced by a hybridoma cell strain which has the preservation number of CCTCC NO: C201504 and is preserved in the China center for type culture collection (CCTCC) on January 29, 2015. The monoclonal antibody specifically neutralizes LMP2A thereby inhibiting tumor cell propagation, reverses induced EMT conversion, inhibits tumor transfer and connects a chemotherapeutic drug and a radiotherapy sensitizer by monoclonal antibody-mediated targeting characteristics so that targeting treatment is realized.

Description

technical field [0001] The invention relates to the fields of biomedicine and immune targeting diagnosis and treatment, in particular to a preparation method and application of an anti-EBV latent membrane protein LMP2A monoclonal antibody. Background technique [0002] Nasopharyngeal carcinoma is one of the common malignant tumors in my country. It occurs in the nasopharyngeal cavity or upper throat, with a high incidence rate, and it occurs most frequently in southern China. 16 to 25 years old and 45 to 54 years old are two age groups where nasopharyngeal carcinoma is prone to occur, and the main targets are young and middle-aged people. The cause of the disease is closely related to EBV infection. The pathological examination of nasopharyngeal carcinoma is mostly poorly differentiated squamous cell carcinoma. The current treatment options are radiotherapy and chemotherapy. However, the five-year survival rate after treatment is about 50% to 60%, and it is prone to recurr...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N5/20G01N33/577G01N33/574
Inventor 徐鑫冯振卿朱进
Owner NANJING MEDICAL UNIV
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