Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer.
Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer.
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DOI：
10.1016/j.jim.2017.04.003
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发表时间：
2017-08
影响因子：
2.2
通讯作者：
Wu Y
中科院分区：
文献类型：
--
作者：
Zhang H;Chan LL;Rice W;Kassam N;Longhi MS;Zhao H;Robson SC;Gao W;Wu Y
杂交瘤筛选是抗体发现的关键步骤，这需要从数百到数千个杂交瘤培养物中迅速识别潜在的克隆，以对抗所需的免疫原。与ELISA和流式细胞术相关的技术问题限制了筛选的准确性，降低了高通量能力，增加了时间和成本。用包被抗原进行常规ELISA筛选对于难以表达的疏水膜抗原或多链蛋白复合物也是不切实际的。在这里，我们展示了采用Celigo图像细胞仪的新型高通量筛选方法，该方法通过直接对比活细胞上的抗体结合信号来避免非特异性信号，无论是否表达重组抗原。通过检测杂交瘤上清液与中国仓鼠卵巢（CHO）细胞表达的重组抗原CD39的结合，优化了基于图像细胞术的高通量筛选方法。接下来，通过连续稀释纯化的CD39抗体来证明图像细胞仪的灵敏度。使用Celigo来测量商业抗体和内部抗体对膜结合CD39的抗体亲和力。这种基于细胞的筛选程序可在一天内完全完成，显著提高了杂交瘤筛选的通量和效率。此外，测量抗体与活细胞的直接结合消除了假阳性和假阴性命中。图像细胞术方法灵敏度高，用途广泛，可以检测到浓度低至~ 5 ng/mL的上清液中的阳性抗体，并同时测定Kd结合亲和系数。我们认为这种筛选方法将极大地促进抗体的发现和筛选技术。
Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ∼5 ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies.
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影响因子：
4.6
作者：
Lee JS;Kallehauge TB;Pedersen LE;Kildegaard HF
通讯作者：
Kildegaard HF
影响因子：
2.2
作者：
Chan, Leo Li-Ying;Smith, Tim;Qiu, Jean
通讯作者：
Qiu, Jean
DOI：
10.1084/jem.20062512
发表时间：
2007-06-11
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Deaglio S;Dwyer KM;Gao W;Friedman D;Usheva A;Erat A;Chen JF;Enjyoji K;Linden J;Oukka M;Kuchroo VK;Strom TB;Robson SC
通讯作者：
Robson SC
影响因子：
7.4
作者：
Sasaki, K;Glass, TR;Ohmura, N
通讯作者：
Ohmura, N
DOI：
10.3390/s131013928
发表时间：
2013-10-15
期刊：
Sensors (Basel, Switzerland)
影响因子：
--
作者：
Long F;Zhu A;Shi H
通讯作者：
Shi H