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Selection of Standards for Bio-Plex Cytokine Assays, Rev A

5. Determine which PMT setting is optimal.
Read the assay on the Bio-Plex array reader after calibrating with the low RP1 target value, then the high value, to determine which setting yields the best results. An assay plate may be read a second time by aspirating the contents of the wells after the first reading and resuspending in 125 l of buffer.


Summary
Bio-Plex assays may be run with a variety of standard curve concentrations. Two sets of concentrations are recommended, a high-sensitivity range for maximum sensitivity or for samples such as serum that fall within the biological range of cytokines, and a broad range for samples where a wide range of cytokine concentrations are expected. The voltage of the reporter PMT may also be varied to maximize assay performance. The high-sensitivity standards should be run following calibration with the high RP1 target value and the broad range standards perform best following calibration with the low RP1 target value. Overall, the highsensitivity standards yield better sensitivity, while maximum dynamic range is achieved using the broad range standards. Excellent assay performance may also be achieved using a unique set of standards if a few simple rules are followed: 1) select a minimum of 8 standard curve concentrations, 2) select a set of concentrations that are evenly distributed across the entire selected range, 3) select a 5PL regression in Bio-Plex Manager software for the most robust assay analysis, 4) prepare the standard curve points using a serial dilution method, and 5) determine which PMT setting is optimal.


References
Baud M, Data analysis, mathematical modeling, pp 656671 in (Mas
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