Development and Validation of Sandwich ELISA Microarrays with Minimal Assay Interference

详细信息    查看全文

• 作者：Rachel M. Gonzalez ; Shannon L. Seurynck-Servoss ; Sheila A. Crowley ; Marty Brown ; Gilbert S. Omenn ; Daniel F. Hayes ; Richard C. Zangar
• 刊名：Journal of Proteome Research
• 出版年：2008
• 出版时间：June 2008
• 年：2008
• 卷：7
• 期：6
• 页码：2406 - 2414
• 全文大小：4672K
• 年卷期：v.7,no.6(June 2008)
• ISSN：1535-3907

文摘

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA’s ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of a multiplexed 24-assay system. We find that nonspecific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a “purified antigen”. We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals than within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.