Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays
基于磁珠分选及固相吸附分析针对HIV-1 gp120核心蛋白的抗体
发布: 2015年10月05日第5卷第20期 DOI: 10.21769/BioProtoc.1614 浏览次数: 6860
评审: Savita NairAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2014
Abstract
Analysis of the functional activity in polyclonal serum following immunization of a complex protein or glycoprotein immunogen is a very important but tedious process. Fine mapping of epitope-specific antibodies is difficult when they are elicited at relatively low levels. In our recent study focused on developing an HIV-1 vaccine, we immunized rabbits with hyperglycosylated stable core immunogens, which were designed using high-resolution structural information to elicit antibodies against the primary receptor-binding, CD4-binding site on HIV-1 gp120. Using a solid phase adsorption assay, we could map the serum antibodies to the conserved CD4-binding site, a known broadly neutralizing determinant on exterior envelope glycoprotein, gp120.
Materials and Reagents
- 15 ml polystyrene tube (BD, Falcon, catalog number: 352096 )
- Dynabeads® MyOneTM Tosylactivated (Thermo Fisher Scientific，Dynal Biotech, catalog number: 65501 )
- Magnetic separator (Thermo Fisher Scientific，Dynal MPC, catalog number: 12301D )
- Bovine serum albumin (BSA) (Biopioneer, catalog number: C0100 )
- Recombinant protein (core gp120) to couple to the beads [expressed and purified in-house. Please check Ingale et al. (2014)]
- Sodium azide (Sigma-Aldrich, catalog number: 438456 )
- DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 10313-021 )
- Fetal Bovine Serum (VWR International, Seradigm, catalog number: 1300-500 )
- Protease inhibitor (Roche Diagnostics, catalog number: 11255500 )
- Glycine-HCl (Sigma-Aldrich, catalog number: G2879 )
- Sodium borate (AMRESCO, catalog number: 0390 )
- Ammonium sulfate, 99+%, ACS reagent (Thermo Fisher Scientific, ACROS ORGANICS, catalog number: 42340-0010 )
- Coupling buffer (see Recipes)
- Blocking buffer (see Recipes)
- Wash buffer (see Recipes)
- Coating buffer (see Recipes)
Equipment
- Magnetic separator (Thermo Fisher Scientific，Dynal MPC, catalog number: 12301D )
- Absorbance reader (Molecular Devices, model: Spectramax Plus )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Ingale, J. and Wyatt, R. T. (2015). Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays. Bio-protocol 5(20): e1614. DOI: 10.21769/BioProtoc.1614.
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分类
免疫学 > 抗体分析 > 抗体检测
微生物学 > 微生物生物化学 > 蛋白质
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