Abstract
The determination of inflammatory cytokine levels is a staple of life science research. Often, sample size does not lend itself to the determination of more than a couple analytes by traditional ELISA methods and performing multiple ELISAs can be time and resource intensive. To this end, several groups have constructed multianalyte fluorescent immunoassays and cytokine microarrays. Seeking to improve on these methodologies, we have constructed an inflammatory cytokine microarray with numerous improvements over current technologies. Our inflammatory cytokine array consists of 12 capture antibodies and appropriate positive and negative controls (printed in triplicate) arrayed on PATH™ thin film nitrocellulose slides. The arrayed anti-analyte antibodies recognize: IL-1α, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFNg, TNFa, MIP-1α, MIP-1β and RANTES. A standard 16 subarray format has been utilized, allowing for the examination of 10 samples on an array slide with a 6 point standard curve. Target analyte is detected through the use of well-characterized, capture matched biotinylated detector antibodies and a high yield neutravidin-linked fluor. With this system, we have observed levels of detection less than 5 pg/ml recombinant standards in a cell culture media. Additionally, natural sample response has been verified by assaying stimulated peripheral blood mononuclear cells and human serum samples.
Published Version
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