Ray Chad A, Bowsher Ronald R, Smith Wendell C, Devanarayan Viswanath, Willey Mark B, Brandt John T, Dean Robert A
Laboratory for Experimental Medicine, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
J Pharm Biomed Anal. 2005 Jan 4;36(5):1037-44. doi: 10.1016/j.jpba.2004.05.024.
Quantification of biomarkers can provide important information about the safety and efficacy of candidate drugs. Unfortunately, limited sample volume and excess costs often limit analysis of multiple biomarkers. We developed, optimized, validated, and implemented a multiplex immunoassay for simultaneous measurement of multiple circulating cytokines: IL-1beta, TNFalpha, IL-6, IL-8, and IL-10. Multiplex immuoassays were performed using the Luminex LabMAP instrument. Capture antibodies for each cytokine were covalently bound to distinct microsphere subsets distinguished by differing dye ratios. The concentration of each individual cytokine determined by measuring orange fluorescence produced by a complex of a biotinylated cytokine-specific antibody and streptavidin-phycoerythrin. The lower limit of quantification for all assays was 20 pg/mL with the exception of IL-8 which was 100 pg/mL. The inter-assay precision was less than 25%CV for all analytes at all control levels both pre-study and in-study. The percent recovery ranged from 83 to 108% pre-study and 90 to 125% in-study. In a linearity assessment, a 15,000 pg/mL multi-analyte control could be diluted 1:50 and maintain expected accuracy. We measured the cytokine concentrations in more than 2000 serum samples from patients with sepsis. Multiplex results for IL-6 were compared to a conventional commercially available ELISA kit. The degree of agreement between the two methods as measured by the concordance correlation coefficient was 84.5%. Multiplex results were 2.36-fold higher than ELISA values on the average. After adjusting for this mean difference, the 95% empirical limits of agreement for the ratio of individual sample values were 0.33, 2.65. This multiplex immunoassay provided simultaneous measurement of circulating cytokines using 80% less patient specimen compared to traditional approaches and at a significantly decreased cost. Efficient use of this platform requires process improvements to fully maximize the positive impact of multiplex assays in clinical drug development.
生物标志物的定量分析可为候选药物的安全性和有效性提供重要信息。遗憾的是，样本量有限和成本过高常常限制了对多种生物标志物的分析。我们开发、优化、验证并实施了一种多重免疫测定法，用于同时检测多种循环细胞因子：白细胞介素-1β（IL-1β）、肿瘤坏死因子α（TNFα）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）和白细胞介素-10（IL-10）。多重免疫测定使用Luminex LabMAP仪器进行。针对每种细胞因子的捕获抗体通过共价结合到由不同染料比例区分的独特微球亚群上。通过测量生物素化的细胞因子特异性抗体与链霉亲和素-藻红蛋白复合物产生的橙色荧光来确定每种细胞因子的浓度。除IL-8的定量下限为100 pg/mL外，所有测定的定量下限均为20 pg/mL。在研究前和研究期间，所有分析物在所有对照水平下的批间精密度均小于25%CV。研究前的回收率范围为83%至108%，研究期间为90%至125%。在线性评估中，15,000 pg/mL的多分析物对照可稀释1:50并保持预期的准确性。我们测量了2000多名脓毒症患者血清样本中的细胞因子浓度。将IL-6的多重检测结果与传统的市售ELISA试剂盒进行比较。通过一致性相关系数测量，两种方法之间的一致程度为84.5%。多重检测结果平均比ELISA值高2.36倍。在调整了这种平均差异后，单个样本值比值的95%经验一致性界限为0.33、2.65。与传统方法相比，这种多重免疫测定法使用的患者样本减少了80%，同时成本显著降低，能够同时检测循环细胞因子。有效利用该平台需要改进流程，以充分发挥多重检测在临床药物开发中的积极作用。
