Efficient discovery of antibody binding pairs using a photobleaching strategy for bead encoding,Lab on a Chip

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Efficient discovery of antibody binding pairs using a photobleaching strategy for bead encoding
Lab on a Chip ( IF 6.1 ) Pub Date : 2024-07-24 , DOI: 10.1039/d4lc00382a
Shira Roth _{1,

2,

3} , Tom Ferrante ₁ , David R Walt _{1,

2,

3}

Affiliation

Dye-encoded bead-based assays are widely used for diagnostics. Multiple bead populations are required for multiplexing and can be produced using different dye colors, labeling levels, or combinations of dye ratios. Ready-to-use multiplex bead populations restrict users to specific targets, are costly, or require specialized instrumentation. In-house methods produce few bead plexes or require many fine-tuning steps. To expand bead encoding strategies, we present a simple, safe, and cost-effective bench-top system for generating bead populations using photobleaching. By photobleaching commercially available dye-encoded magnetic beads for different durations, we produce three times as many differentiable bead populations on flow cytometry from a single dye color. Our photobleaching system uses a high-power LED module connected to a light concentrator and a heat sink. The beads are photobleached in solution homogeneously by constant mixing. We demonstrate this photobleaching method can be utilized for cross-testing antibodies, which is the first step in developing immunoassays. The assay uses multiple photobleached encoded beads conjugated with capture antibodies to test many binding pairs simultaneously. To further expand the number of antibodies that can be tested at once, several antibodies were conjugated to the same bead, forming a pooled assay. Our assay predicts the performance of antibody pairs used in ultrasensitive Simoa assays, narrowing the number of cross-tested pairs that need to be tested by at least two-thirds and, therefore, providing a rapid alternative for an initial antibody pair screening. The photobleaching system can be utilized for other applications, such as multiplexing, and for photobleaching other particles in solution.

中文翻译：

使用珠子编码的光漂白策略有效发现抗体结合对

基于染料编码珠的检测广泛用于诊断。多重检测需要多个珠群，并且可以使用不同的染料颜色、标记水平或染料比例的组合来产生。即用型多重微珠群将用户限制于特定目标，成本高昂，或者需要专门的仪器。内部方法产生很少的珠复合物或需要许多微调步骤。为了扩展珠子编码策略，我们提出了一种简单、安全且经济高效的台式系统，用于使用光漂白生成珠子群。通过对市售染料编码磁珠进行不同持续时间的光漂白，我们在流式细胞术中用单一染料颜色产生了三倍的可区分磁珠群。我们的光漂白系统使用连接到聚光器和散热器的高功率 LED 模块。通过不断混合，珠子在溶液中均匀地光漂白。我们证明这种光漂白方法可用于交叉测试抗体，这是开发免疫测定的第一步。该测定使用多个与捕获抗体缀合的光漂白编码珠来同时测试许多结合对。为了进一步扩大可同时测试的抗体数量，将几种抗体缀合到同一个珠子上，形成混合测定。我们的测定预测了超灵敏 Simoa 测定中使用的抗体对的性能，将需要测试的交叉测试对的数量减少了至少三分之二，因此为初始抗体对筛选提供了一种快速替代方案。光漂白系统可用于其他应用，例如多重分析，以及用于光漂白溶液中的其他颗粒。

更新日期：2024-07-24

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