title: Development and validation of sandwich ELISA microarrays with minimal assay interference.
abstract: 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.
authors.affiliation: Pacific Northwest National Laboratory, 902 Battelle Boulevard, P7-56, Richland, Washington 99354, USA.
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Gonzalez
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Seurynck-Servoss
authors.forename: Sheila A
authors.initials: SA
authors.lastname: Crowley
authors.forename: Marty
authors.initials: M
authors.lastname: Brown
authors.forename: Gilbert S
authors.initials: GS
authors.lastname: Omenn
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Hayes
authors.forename: Richard C
authors.initials: RC
authors.lastname: Zangar
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000032:analysis / Q000276:immunology; D018931:Antineoplastic Agents, Hormonal / Q000627:therapeutic use; D001798:Blood Proteins / Q000032:analysis / Q000187:drug effects / Q000276:immunology; D001943:Breast Neoplasms / Q000097:blood / Q000188:drug therapy; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004815:Epidermal Growth Factor / Q000097:blood; D005260:Female; D006801:Humans; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000276:immunology; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013629:Tamoxifen / Q000627:therapeutic use
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D018931:Antineoplastic Agents, Hormonal; D001798:Blood Proteins; D011506:Proteins; D013629:Tamoxifen; D004815:Epidermal Growth Factor