Screening one bead one compound libraries against serum using a flow cytometer: Determination of the minimum antibody concentration required for ligand discovery.
Screening one bead one compound libraries against serum using a flow cytometer: Determination of the minimum antibody concentration required for ligand discovery.
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DOI：
10.1016/j.bmcl.2018.01.033
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发表时间：
2018-09-01
影响因子：
2.7
通讯作者：
Kodadek T
中科院分区：
文献类型：
--
作者：
Erharuyi O;Simanski S;McEnaney PJ;Kodadek T
可以针对血清样品筛选一珠一化合物（OBOC）文库以鉴定该混合物中抗体的配体。在该方案中，通过用荧光标记的二抗染色来鉴定命中珠。当针对两组不同的血清进行筛选时，可以发现区分两个群体的抗体和它们的配体。DNA编码技术在OBOC文库中的应用允许使用10 μm的珠粒进行文库制备和筛选，其通过标准流式细胞仪，允许荧光命中珠粒容易地与展示非配体的珠粒分离。使用这种方法发现抗体生物标志物的一个重要问题是其分析灵敏度。换句话说，IgG的丰度必须达到多少才能在流式细胞仪的无偏筛选中从血清中提取出来？我们在这里报告一个模型研究，其中单克隆抗体与已知的不同亲和力的配体掺杂到血清中。我们发现，对于从无偏组合文库中分离的典型抗体配体，靶抗体必须以10-50 nM存在。真正的抗原，其结合具有显着更高的亲和力，可以检测到丰度低得多的血清抗体。
One bead one compound (OBOC) libraries can be screened against serum samples to identify ligands to antibodies in this mixture. In this protocol, hit beads are identified by staining with a fluorescent labeled secondary antibody. When screens are conducted against two different sets of serum, antibodies, and ligands to them, can be discovered that distinguish the two populations. The application of DNA-encoding technology to OBOC libraries has allowed the use of 10 μm beads for library preparation and screening, which pass through a standard flow cytometer, allowing the fluorescent hit beads to be separated from beads displaying non-ligands easily. An important issue in using this approach for the discovery of antibody biomarkers is its analytical sensitivity. In other words, how abundant must an IgG be to allow it to be pulled out of serum in an unbiased screen using a flow cytometer? We report here a model study in which monoclonal antibodies with known ligands of varying affinities are doped into serum. We find that for antibody ligands typical of what one isolates from an unbiased combinatorial library, the target antibody must be present at 10-50 nM. True antigens, which bind with significantly higher affinity, can detect much less abundant serum antibodies.
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影响因子：
15
作者：
Udugamasooriya, D. Gomika;Dineen, Sean P.;Kodadek, Thomas
通讯作者：
Kodadek, Thomas
影响因子：
3.2
作者：
Olivos, HI;Bachhawat-Sikder, K;Kodadek, T
通讯作者：
Kodadek, T
影响因子：
4.8
作者：
Sarkar, Mohosin;Liu, Yun;Kodadek, Thomas
通讯作者：
Kodadek, Thomas
影响因子：
64.5
作者：
Reddy MM;Wilson R;Wilson J;Connell S;Gocke A;Hynan L;German D;Kodadek T
通讯作者：
Kodadek T
影响因子：
15
作者：
Doran TM;Sarkar M;Kodadek T
通讯作者：
Kodadek T