一种比较抗体染色试剂亮度和估计每个细胞结合抗体的定量方法

Q1 Health Professions Current Protocols in Cytometry Pub Date : 2016-07-01 DOI:10.1002/cpcy.6
Aaron B. Kantor, Wayne A. Moore, Stephen Meehan, David R. Parks
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引用次数: 10

摘要

我们提出了一种定量的方法来比较抗体染料试剂的亮度和估计每个细胞结合的抗体。该方法基于测试试剂和填充试剂与抗体捕获微球的互补结合。几个等分的抗体捕获珠被不同数量的测试偶联物染色。然后用含有不同荧光团的第二种共轭物填充珠上剩余的结合位点。最后,与填充共轭物相比,测试共轭物的荧光用于测量测试共轭物的相对亮度。test-fill方法的基本假设是,如果需要一个测试抗体的X分子来降低Y个单位的填充信号,则需要任何其他测试抗体的相同X分子来产生相同的效果。我们应用二次拟合来评估不同数量的测试试剂的测试填充信号关系。如果拟合接近线性,我们认为该测试试剂适合用于抗体结合的定量评价。为了校准每个抗体珠结合的抗体,使用每个抗体1个PE分子的PE偶联物作为测试试剂,荧光刻度用Quantibrite PE珠校准。当每个抗体分子的荧光被确定为特定的偶联物时,该偶联物可用于测量每个细胞结合的抗体。这提供了不同的共轭亮度的比较,当进行仪器的统计光电子(Spe)尺度是已知的。©2016 by John Wiley &儿子,Inc。
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A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell

We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

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Current Protocols in Cytometry
Current Protocols in Cytometry Health Professions-Medical Laboratory Technology
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期刊介绍: Published in affiliation with the International Society for Advancement of Cytometry, Current Protocols in Cytometry is a "best practices" collection that distills and organizes the absolute latest techniques from the top cytometry labs and specialists worldwide. It is the most complete set of peer-reviewed protocols for flow and image cytometry available.
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