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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   

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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

  1. 15 ml polystyrene tube (BD, Falcon, catalog number: 352096 )
  2. Dynabeads® MyOneTM Tosylactivated (Thermo Fisher Scientific,Dynal Biotech, catalog number: 65501 )
  3. Magnetic separator (Thermo Fisher Scientific,Dynal MPC, catalog number: 12301D )
  4. Bovine serum albumin (BSA) (Biopioneer, catalog number: C0100 )
  5. Recombinant protein (core gp120) to couple to the beads [expressed and purified in-house. Please check Ingale et al. (2014)]
  6. Sodium azide (Sigma-Aldrich, catalog number: 438456 )
  7. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 10313-021 )
  8. Fetal Bovine Serum (VWR International, Seradigm, catalog number: 1300-500 )
  9. Protease inhibitor (Roche Diagnostics, catalog number: 11255500 )
  10. Glycine-HCl (Sigma-Aldrich, catalog number: G2879 )
  11. Sodium borate (AMRESCO, catalog number: 0390 )
  12. Ammonium sulfate, 99+%, ACS reagent (Thermo Fisher Scientific, ACROS ORGANICS, catalog number: 42340-0010 )
  13. Coupling buffer (see Recipes)
  14. Blocking buffer (see Recipes)
  15. Wash buffer (see Recipes)
  16. Coating buffer (see Recipes)

Equipment

  1. Magnetic separator (Thermo Fisher Scientific,Dynal MPC, catalog number: 12301D )
  2. Absorbance reader (Molecular Devices, model: Spectramax Plus )

Procedure

Dynabeads MyOne Tosylactivated beads are superparamagnetic, polystyrene beads with polyurethane layer designed for bio-magnetic separations. These beads were freshly tosyl-activated, coupled with selected core gp120 variants. We used the core-conjugated Dynal beads to fractionate site-specific antibodies from polyclonal anti-sera elicited by HIV-1 core immunization. Anti-sera were adsorbed over isogenic glycoprotein antigens possessing epitope-specific mutations that were covalently attached to the Dynabeads. Following this adsorption method we were able to enrich and detect antibodies elicited against the desired epitope.

  1. Conjugation of protein/glycoprotein to the Dynabeads
    1. Tosyl-activated paramagnetic Dynabeads were washed by transferring beads to a tube placed on a magnet until the beads migrated to the side of the tube and the liquid was clear. The tube was removed from the magnet and beads were resuspended in 1 ml of coating buffer. The tube was again placed on the magnet and the liquid removed. These beads were then used as described below.
    2. 2 mg protein (at a concentration of >5 mg/ml) was mixed with 100 mg (1 ml, initial volume before washing the beads) tosyl-activated paramagnetic Dynabeads in a total volume of 2.5 ml of coupling buffer with slow tilt rotation at 37 °C for 16-to-24 h in a 15 ml polystyrene tube.
    3. The tube was inserted into the magnetic separator for 2 min to separate the glycoprotein-conjugated beads from buffer containing unconjugated, free protein. Non-covalently linked protein was discarded.
    4. The Dynabeads were resuspended in 1.25 ml blocking buffer at 37 °C for additional 16-to-24 h.
    5. The Dynabeads were washed 3 times with 2 ml wash buffer at room temperature and stored at 4 °C in 500 μl PBS containing 0.02% sodium azide and protease inhibitor (1 tablet in 50 ml). Beads were separated between each wash on the magnetic separator.

  2. Solid phase adsorption
    1. Protein-coupled Dynabeads were washed three times with 2 ml DMEM containing 10% FBS and incubated for 30 min at RT to block nonspecific binding sites on the beads. Between every wash beads were separated using the magnetic separator.
    2. Rabbit sera derived from the same group of animals were pooled and diluted 40-fold with DMEM. Eighty μl of the diluted sera was added to 100 μl of protein-conjugated beads and incubated for additional 30 min with gentle rocking at room temperature. Rabbit sera were obtained from a separate immunization experiment, where rabbits were immunized with HIV-1 core glycoptoteins.
    3. Un-bound serum antibodies were separated from the protein-conjugated beads using a magnet.
    4. Multiple rounds of adsorption were performed. Fresh protein-coupled Dynabeads were used for each round of adsorption for complete removal of specific antibodies from the diluted serum to the solid phase.
    5.  For each set of serum adsorptions, blank beads, (same amounts as the experimental beads) devoid of protein were used as negative controls.
    6. To check for the complete enrichment of core- and site-specific antibodies, serum IgGs were eluted from the beads by addition of 100 mM Glycine-HCl (pH 2.7). In parallel, the serum fractions containing un-bound antibodies were also analyzed. ELISAs were performed using wild-type and site-specific mutant proteins as targets on the ELISA plate as described in Ingale et al. (2014).

Recipes

  1. Coupling buffer
    0.1 M sodium borate (pH 9.5) with 1 M ammonium sulfate
  2. Blocking buffer
    PBS (pH 7.4) with 0.5% (wt/vol) BSA and 0.05% Tween-20
  3. Wash buffer
    PBS (pH 7.4) with 0.1% (wt/vol) BSA and 0.05% Tween-20
  4. Coating buffer
    0.1 M sodium borate (pH 9.5)

Acknowledgments

This protocol was previously used in Ingale et al. (2014).

References

  1. Ingale, J., Tran, K., Kong, L., Dey, B., McKee, K., Schief, W., Kwong, P. D., Mascola, J. R. and Wyatt, R. T. (2014). Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies. J Virol 88(24): 14002-14016.

简介

我们使用"微汤姆"来研究番茄果实成熟和发育机制。 "微汤姆"适合于培养和实验,由于其小尺寸10至20厘米的高度和短的生命周期3个月。 还有大量关于"Micro-Tom"的公开信息,包括EST,全长cDNA克隆和转录组数据。 "Micro-Tom"植物在水培培养基中在荧光下使用在温室或植物室中的拟南芥文化架来生长,以获得具有转录组和蛋白质组分析的可重复性的数据。...

材料和试剂

  1. 15ml聚苯乙烯管(BD,Falcon,目录号:352096)
  2. (Thermo Fisher Scientific,Dynal Biotech,目录号:65501)。
  3. 磁性分离器(Thermo Fisher Scientific,Dynal MPC,目录号:12301D)
  4. 牛血清白蛋白(BSA)(Biopioneer,目录号:C0100)
  5. 重组蛋白(核心gp120)偶联到珠[表达和内部纯化。请检查Ingale 等人(2014)]
  6. 叠氮化钠(Sigma-Aldrich,目录号:438456)
  7. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:10313-021)
  8. 胎牛血清(VWR International,Seradigm,目录号:1300-500)
  9. 蛋白酶抑制剂(Roche Diagnostics,目录号:11255500)
  10. 甘氨酸-HCl(Sigma-Aldrich,目录号:G2879)
  11. 硼酸钠(AMRESCO,目录号:0390)
  12. 硫酸铵,99 +%,ACS试剂(Thermo Fisher Scientific,ACROS ORGANICS,目录号:42340-0010)
  13. 耦合缓冲区(参见配方)
  14. 阻止缓冲区(参见配方)
  15. 洗涤缓冲液(见配方)
  16. 涂层缓冲液(见配方)

设备

  1. 磁性分离器(Thermo Fisher Scientific,Dynal MPC,目录号:12301D)
  2. 吸光度读数器(Molecular Devices,型号:Spectramax Plus)

程序

Dynabeads MyOne Tosylactivated珠是超顺磁,聚苯乙烯珠与聚氨酯层设计用于生物磁性分离。这些珠子是新鲜甲苯磺酰基活化的,与选定的核心gp120变体偶联。我们使用核心结合的Dynal珠分离从HIV-1核心免疫引起的多克隆抗血清的位点特异性抗体。抗血清吸附在具有共价连接到Dynabeads的表位特异性突变的同基因糖蛋白抗原上。根据这种吸附方法,我们能够富集和检测针对所需表位引发的抗体。

  1. 蛋白/糖蛋白与Dynabeads的结合
    1. 甲硅烷基活化的顺磁Dynabeads通过转移珠洗涤 ?到放置在磁体上的管,直到小珠移动到侧面 管和液体是澄清的。从磁体上取下管 将珠子重悬于1ml包被缓冲液中。再次放置管 在磁铁上除去液体。然后将这些珠用作
    2. 2mg蛋白质(浓度> 5mg/ml) ?与100mg(1ml,洗涤珠子之前的初始体积)混合, 甲苯磺酰基活化的顺磁Dynabeads在总体积为2.5ml 偶联缓冲液,在37℃下缓慢倾斜旋转16至24小时 ml聚苯乙烯管。
    3. 将管插入磁体中 分离器中2分钟以从中分离糖蛋白缀合的珠子 缓冲液中含有未结合的游离蛋白。非共价连接 蛋白质被丢弃。
    4. 将Dynabeads在37℃下再悬浮于1.25ml封闭缓冲液中另外16至24小时。
    5. Dynabeads在室温下用2ml洗涤缓冲液洗涤3次 并在4℃下在含有0.02%钠的500μlPBS中储存 叠氮化物和蛋白酶抑制剂(1片在50ml中)。珠分离 在每次洗涤之间在磁选机上。

  2. 固相吸附
    1. 蛋白偶联的Dynabeads用2ml DMEM洗涤三次 含有10%FBS并在室温下孵育30分钟以阻断非特异性 珠上的结合位点。在每个洗涤珠之间分离 使用磁选机
    2. 来自相同组的兔血清 ?合并动物,并用DMEM稀释40倍。八十微升 将稀释的血清加入到100μl蛋白质缀合的珠中 在室温下温和摇动孵育另外30分钟。 ?兔血清获自单独的免疫实验, 其中兔子用HIV-1核心细胞凋亡蛋白免疫
    3. 使用磁铁将未结合的血清抗体与蛋白质缀合的珠分离
    4. 进行多轮吸附。新鲜蛋白偶联 Dynabeads用于每轮吸附以完全除去 ?特异性抗体从稀释的血清至固相
    5.  对于每组血清吸附,将空白珠(相同量的 无实验珠)用作阴性对照。
    6. 检查核心和位点特异性的完全富集 抗体,通过加入100mM从珠子洗脱血清IgG 甘氨酸-HCl(pH 2.7)。平行地,含有血清级分 还分析了未结合的抗体。使用进行ELISA 野生型和位点特异性突变蛋白作为ELISA上的靶标 如在Ingale et al。(2014)中所述。

食谱

  1. 耦合缓冲区
    0.1M硼酸钠(pH9.5)与1M硫酸铵混合
  2. 阻塞缓冲区
    含有0.5%(wt/vol)BSA和0.05%Tween-20的PBS(pH7.4)
  3. 洗涤缓冲液
    具有0.1%(wt/vol)BSA和0.05%Tween-20的PBS(pH 7.4)
  4. 包被缓冲液
    0.1M硼酸钠(pH9.5)

致谢

此协议以前用于Ingale等人(2014)。

参考文献

  1. Ingale,J.,Tran,K.,Kong,L.,Dey,B.,McKee,K.,Schief,W.,Kwong,P.D.,Mascola,J.R.and Wyatt,R.T。 设计用于呈递CD4结合位点的高糖基化稳定核心免疫原优先被广泛中和抗体识别。 a> J Virol 88(24):14002-14016。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
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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