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Hybridoma cell strain, CD68 monoclonal antibody, preparation method and application

A hybridoma cell line and monoclonal antibody technology, applied in the field of biomedicine, can solve the problems of poor stability and high cost of monoclonal antibody preparation, and achieve the effects of high accuracy, high specificity and sensitivity, and clear staining background.

Active Publication Date: 2018-07-27
富恩生物技术(成都)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most CD68 antibodies at home and abroad are mainly polyclonal antibodies, and the preparation cost of monoclonal antibodies is high and the stability is poor. Important for diagnosis and scientific research

Method used

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  • Hybridoma cell strain, CD68 monoclonal antibody, preparation method and application
  • Hybridoma cell strain, CD68 monoclonal antibody, preparation method and application
  • Hybridoma cell strain, CD68 monoclonal antibody, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of CD68 Monoclonal Antibody

[0039] 1) Immunogen preparation

[0040] Using eukaryotically expressed CD68 protein as the immunogen, the eukaryotically expressed CD68 protein is CD68 protein expressed by human engineered cells (purchased from Beijing Shenzhou Yiqiao, article number: 11192-H08B1), immunized female Balb / c mice, and obtained spleen lymph Cells were used for hybridoma fusion experiments.

[0041] 2) Immunization of Balb / c mice with immunogen

[0042] The purified recombinant CD68 protein was dissolved and diluted to 1.0 mg / mL with 10 mmol / LPBS of pH 7.4 after measuring the protein content. Seven female Balb / c mice aged 6-8 weeks and with equal body weight were selected. For the first immunization, CD68 protein was emulsified and mixed with an equal volume of Freund's complete adjuvant as the immunogen; the second and third immunizations were performed every two weeks, and the adjuvant was changed to Freund's incomplete adjuvant; 3 d...

Embodiment 2

[0069] Example 2 Screening of the best CD68 monoclonal antibody combined with CD68 antigen

[0070] The technical scheme of immunohistochemistry (enzyme-labeled antibody indirect method), the obtained group of CD68 antibodies is used to immunohistochemically stain the serial sections of the same tissue under the same conditions, and the antibody with the strongest specificity and highest titer is screened out of the antibodies , and detect the expression of the CD68 monoclonal antibody of the present invention on the tumor tissue.

[0071] 1) Paraffin sections of giant cell tumor tissue were routinely dewaxed and hydrated: the sections were baked at 65°C for 30 minutes, soaked in xylene for 15 minutes×2 times; soaked in absolute ethanol for 10 minutes×2 times; Soak in % alcohol, 85% alcohol, 75% alcohol for 5 minutes each; soak in distilled water for 5 minutes x 3 times;

[0072] 2) Antigen retrieval:

[0073] Add 500mL citric acid repair solution (pH6.0) into the stainless ...

Embodiment 3

[0089] Example 3 Comparing CD68 Monoclonal Antibody Immunohistochemical Detection Specificity

[0090] Choose to purchase CD68 monoclonal antibody from Beijing Zhongshan in China, use the manual and explore the best conditions for 17E11, stain the same tumor tissue sections respectively, compare the staining conditions, and determine whether the specificity is consistent. The specific operation is as follows:

[0091] 1) Paraffin tissue sections of giant cell tumor of bone, colorectal cancer, and lung cancer tissue were routinely dewaxed and hydrated: the sections were baked in an incubator at 65°C for 30 minutes, soaked in xylene for 15 minutes, twice; Soak in absolute ethanol for 10 minutes, twice; soak in 95% alcohol, 85% alcohol, and 75% alcohol for 5 minutes each; soak in distilled water for 5 minutes x 3 times;

[0092] 2) Antigen restoration: 0.01M citric acid restoration solution (pH6.0), high-pressure restoration for 2min30s; soaking in distilled water for 5min×3 time...

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Abstract

The invention relates to a hybridoma cell strain, a CD68 monoclonal antibody, a preparation method and application. The preparation method of the monoclonal antibody comprises the following steps: taking purified CD68 recombinant extracellular proteins as immunogens for immunizing animal bodies; fusing myeloma cells SP2 / 0 with B lymphocytes of the immunized animal bodies to obtain hybridoma cells;screening specific hybridoma cell positive clones, carrying out cell cloning on the positive clones, screening hybridoma cells capable of stably secreting CD68 monoclonal antibodies to obtain the CD68 monoclonal antibody. The CD68 monoclonal antibody has the characteristics of high specificity, high affinity, high stability and high titer.

Description

technical field [0001] The present invention relates to the field of biomedical technology, and more specifically, relates to a hybridoma cell line capable of producing CD68 monoclonal antibody, CD68 monoclonal antibody, and the preparation method and application of the antibody. Background technique [0002] CD68 is a 110KDa glycoprotein associated with lysosomal granules. CD68 is a marker of macrophages in many human tissues, including bone marrow, spleen, intestinal lamina propria and alveoli. Peripheral blood monocytes are also positive, while bone marrow precursors and peripheral blood granulocytes can also be studied. In recent years, it has been reported in the literature that CD68 can also be used as a marker of histiocytes, mainly for the study of macrophages, acute and chronic myeloid leukemia, and tumors derived from histiocytes such as malignant fibrous histiocytoma. [0003] With the deepening of tumor immunology research, it is known that the cells infiltrati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/28G01N33/577G01N33/574
CPCC07K16/2896C12N5/163G01N33/57492G01N33/577G01N2333/70596
Inventor 蔡瑞丽何荫良李裕萍颜先强
Owner 富恩生物技术(成都)有限公司
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