Departments of Medicine, Biomedical Engineering, Biological Chemistry, Bayview Proteomics Center, Johns Hopkins University, Baltimore, MD 21224, USA.
Clin Chem. 2010 Feb;56(2):314-8. doi: 10.1373/clinchem.2009.135087. Epub 2009 Dec 18.
Candidate biomarkers discovered with high-throughput proteomic techniques (along with many biomarkers reported in the literature) must be rigorously validated. The simultaneous quantitative assessment of multiple potential biomarkers across large cohorts presents a major challenge to the field. Multiplex immunoassays represent a promising solution, with the potential to provide quantitative data via parallel analyses. These assays also require substantially less sample and reagents than the traditional ELISA (which is further limited by its ability to measure only a single antigen). We have measured the reproducibility, reliability, robustness, accuracy, and throughput of commercially available multiplex immunoassays to ascertain their suitability for serum biomarker analysis and validation.
Assay platforms MULTI-ARRAY (Meso Scale Discovery), Bio-Plex (Bio-Rad Laboratories), A(2) (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) were selected as representative examples of technologies currently used for high-throughput immunoanalysis. All assays were performed according to protocols specified by the manufacturers and with the reagents (diluents, calibrators, blocking reagents, and detecting-antibody mixtures) included with their kits.
The quantifiable interval determined for each assay and antigen was based on precision (CV < 25%) and percentage recovery (measured concentration within 20% of the actual concentration). The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY system had the most linear signal output over the widest concentration range (10(5) to 10(6)). Cytokine concentrations in unspiked and cytokine-spiked serum samples from healthy individuals were further investigated with the MULTI-ARRAY and Bio-Plex assays.
The MULTI-ARRAY and Bio-Plex multiplex immunoassay systems are the most suitable for biomarker analysis or quantification.
高通量蛋白质组学技术发现的候选生物标志物（以及文献中报道的许多生物标志物）必须经过严格验证。在大型队列中同时对多个潜在生物标志物进行定量评估对该领域提出了重大挑战。多重免疫分析代表了一种很有前途的解决方案，具有通过平行分析提供定量数据的潜力。与传统 ELISA 相比，这些检测所需的样本和试剂要少得多（传统 ELISA 由于只能测量单一抗原，其应用也受到限制）。我们已经测量了市售多重免疫分析的重现性、可靠性、稳健性、准确性和通量，以确定它们是否适合血清生物标志物分析和验证。
选择 Meso Scale Discovery（Meso Scale Discovery，Meso Scale Discovery）的 MULTI-ARRAY 平台、Bio-Rad Laboratories（Bio-Rad Laboratories）的 Bio-Plex 平台、Beckman Coulter（Beckman Coulter）的 A(2) 平台、Whatman Schleicher & Schuell BioScience（Whatman Schleicher & Schuell BioScience）的 FAST Quant 平台和 Bender MedSystems（Bender MedSystems）的 FlowCytomix 平台作为目前用于高通量免疫分析的代表性技术。所有检测均按照制造商的方案进行，使用试剂盒中包含的试剂（稀释剂、校准品、封闭试剂和检测抗体混合物）进行。
根据精密度（CV<25%）和回收率（测量浓度与实际浓度的偏差在 20%以内）确定了每个检测和抗原的可定量区间。MULTI-ARRAY 和 Bio-Plex 检测具有最佳的性能，检测限最低，MULTI-ARRAY 系统在最宽的浓度范围内具有最线性的信号输出（10(5) 至 10(6)）。利用 MULTI-ARRAY 和 Bio-Plex 检测进一步研究了健康个体未加标和加标血清样本中的细胞因子浓度。
MULTI-ARRAY 和 Bio-Plex 多重免疫分析系统最适合用于生物标志物分析或定量。
