Antibody-based protein multiplex platforms: technical and operational challenges.
Antibody-based protein multiplex platforms: technical and operational challenges.
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DOI：
10.1373/clinchem.2009.127514
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发表时间：
2010-02
影响因子：
9.3
通讯作者：
Klee GG
中科院分区：
文献类型：
--
作者：
Ellington AA;Kullo IJ;Bailey KR;Klee GG
多种蛋白质生物标志物的测量可以改善临床环境中的风险分层。这一概念刺激了多重免疫分析平台的发展，该平台提供对同一样本的多个平行蛋白质测量。我们提供了基于抗体的多重免疫分析平台的概述，并讨论了技术和操作挑战。多重免疫测定使用传统的免疫测定原理，其中高亲和力捕获配体以平面形式或悬浮液中的微球固定在平行阵列中。多重免疫测定的开发需要对测定配置和分析性能进行严格验证，以最大限度地减少测定的不精密度和不准确度。与多重配置相关的挑战包括捕获配体的选择和固定、校准、抗体和蛋白质与测定稀释剂之间的干扰以及测定定量限的兼容性。我们讨论了这些挑战的潜在解决方案。用于评估分析性多重测定性能的标准包括线性范围、分析专属性、回收率以及与质量参考方法的比较。用于多重蛋白质免疫测定的质量控制材料尚未开发完善，并且需要用于解释多重质量控制数据的算法。技术和操作挑战阻碍了在临床环境中实施多重测定。在体外诊断市场上广泛应用多重阵列之前，需要指导多重测定配置、分析验证和质量控制的正式程序。
The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen. We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed. Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.