Detection of Tumor Cell Surface-reactive Antibodies
肿瘤细胞膜结合性抗体的检测
发布: 2014年05月05日第4卷第9期 DOI: 10.21769/BioProtoc.1114 浏览次数: 8329
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参见作者原研究论文
The authors used this protocol in:
May 2013
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Abstract
Vaccine-based immunotherapy is being used to treat dogs with primary brain tumors. The vaccines are composed of a lysate of autologous tumor cells, which stimulate an immune response producing tumor specific antibodies that are capable of inducing antibody-dependent cell-mediated cytotoxicity to allogeneic, as well as autologous, tumor cells. This protocol will describe the tumor cell serum antibody-binding assay to measure the tumor-reactive IgG antibody response. Key features of this assay are that it is performed with sera collected from the canine patient prior to and following vaccination as the source of antibodies and canine brain tumor cells used as the target cells.
Materials and Reagents
- Tumor cells
Note: Tumor sample is harvested from pet dogs during brain surgery.
- BD MatrigelTM (BD Bioscience, catalog number: 354230 )
- DMEM/F12 Medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11320-033 )
- 50x B-27 supplement without vitamin A (Life Technologies, Gibco®, catalog number: 12587-010 )
- 100x N2 supplement (Life Technologies, Gibco®, catalog number: 17502-048 )
- Recombinant human Epidermal Growth Factor (Pepro Tech, catalog number: AF-100-15 )
- Recombinant human fibroblast growth factor-basic (Pepro Tech, catalog number: 100-18B )
- 50x Penicillin-streptomycin solution (Mediatech, Cellgro®, catalog number: 30-001-CI )
- Dispase (BD Bioscience, catalog number: 354235 )
- 1x Phosphate-Buffered Saline (Mediatech, Cellgro®, catalog number: 21-040-CV )
- MACS BSA stock solution (10% BSA) (Miltenyi Biotec, catalog number: 130-091-376 )
- 1x TrypLETM Express (Life Technologies, Gibco®, catalog number: 12605-010 )
- 0.4% Trypan Blue Solution (Life Technologies, Gibco®, catalog number: 15250-061 )
- Goat anti-canine IgG (H&L) f(ab’)2-fluorescein isothiocyanate (American Qualex, catalog number: F145FN )
- Neural Stem Cell (NSC) medium (see Recipes)
- Matrigel Coating solution (see Recipes)
- rh-EGF stock solution (see Recipes)
- rh-FGF basic stock solution (see Recipes)
Equipment
- 10 mm culture plates (Sigma-Aldrich, Corning® Costar®, catalog number: CLS430165 )
- Scalpel blade
- 100 μm filter (Corning Incorporated, catalog number: 352360 )
- BD FACS tubes (12 x75 mm) (BD Biosciences, Falcon®, catalog number: 352235 )
- 15 ml tubes (BD Biosciences, Falcon®, catalog number: 352096 )
- Culture dish (100 x 20 mm) (Sarstedt AG, catalog number: 83.1802 )
- 1.5 ml Eppendorf microcentrifuge tubes (Sigma-Aldrich, catalog number: T9661-500EA )
- Neubauer hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
- Incubator (5% CO2, 5% O2, 37 °C) (BioSpherix)
- Shaker table (Eppendorf, New Brunswick Excella® E25 incubator shaker)
- Centrifuge (Thermo Fisher Scientific, model: ST40R )
- Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17 )
- BD FACSCanto three-laser flow cytometer (BD)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Andersen, B. M., Goulart, M. R., Olin, M. R. and Pluhar, G. E. (2014). Detection of Tumor Cell Surface-reactive Antibodies. Bio-protocol 4(9): e1114. DOI: 10.21769/BioProtoc.1114.
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分类
癌症生物学 > 肿瘤免疫学 > 免疫学试验
癌症生物学 > 通用技术 > 生物化学试验
免疫学 > 抗体分析 > 抗体功能
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