Functional single-cell hybridoma screening using droplet-based microfluidics
Functional single-cell hybridoma screening using droplet-based microfluidics
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DOI：
10.1073/pnas.1204514109
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发表时间：
2012-07-17
影响因子：
11.1
通讯作者：
Merten, Christoph A.
中科院分区：
文献类型：
--
作者：
El Debs, Bachir;Utharala, Ramesh;Merten, Christoph A.
单克隆抗体可以特异性结合甚至抑制药物靶点，因此已成为发展最快的一类人类治疗药物。虽然可以使用噬菌体展示等系统以非常高的通量筛选它们的结合亲和力，但筛选功能特性（例如，例如，在一个实施例中，药物靶标的抑制）更具挑战性。通常，这些筛选需要产生永生化杂交瘤细胞，以及在微量滴定板中进行数周的克隆扩增，并且可以测定的克隆数通常不超过几千个。我们在这里提出了一个微流控平台，允许在不到一天的时间内对多达300，000个单个杂交瘤细胞克隆进行功能筛选。这种方法也应该适用于非永生化的原代B细胞，因为不需要细胞增殖：将单个细胞包封到水性微滴中，并基于荧光直接测定抑制药物靶标的抗体的释放。我们使用该系统进行了抑制血管紧张素转换酶1的抗体的模型筛选，血管紧张素转换酶1是高血压和充血性心力衰竭药物的靶标。当将表达这些抗体的细胞以1：10，000的比例掺入不相关的杂交瘤细胞群中时，我们观察到荧光激活液滴分选后的9，400倍富集。观察到单个杂交瘤系内单细胞水平的抗体表达水平的广泛变化，并且可以成功地分选和再培养高表达子。
Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e. g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.