The broad dynamic ranges of the Lumit™ Cytokine Immunoassays enable direct measurements of released cytokine without having to dilute samples. When titrating an inducer or an inhibitor, this results in greater capture of data without having to dilute different concentrations of the test agent to ensure the values fall within the standard curve range.
For the LPS-primed J774A.1 cells treated with nigericin, the amount of IL-1β released from 5 × 104 cells/well led to concentrations that would have been beyond the ELISA standard curve range (see Figure 1), complicating the assay setup since wells with lower MCC950 concentrations would have required dilution to be within the ELISA dynamic range. The Lumit™ Immunoassay enabled quantifying released IL-1β from a very broad MCC950 inhibitor range of 10µM down to 5pM without having to dilute any of the samples (Figure 5). The broad dynamic range captured the highest levels of IL-1β from uninhibited cells and the sensitivity detected the lowest levels of IL-1β from nearly completely inhibited cells.
A comparison of different nonlinear regression curve fitting and interpolation methods indicated that the 2nd polynomial method was a better fit for the broad calibration curve of the Lumit™ IL-1β (Mouse) Immunoassay compared to 4PL or 3rd polynomial, and likewise, the 2nd polynomial method was better able to interpolate values over the entire dynamic range, compared to the other two methods. The IC50s were similar for 2nd and 3rd polynomial, at 97 and 98nM respectively, but different from the 4PL calculated IC50 of 142nM. The limitations of the 4PL interpolation would indicate the higher IC50 is not accurate.
The GloMax® Discover System enables very rapid interpolation of unknown cytokine concentrations with built-in programs and convenient displays of interpolated values right in the same well as the RLU value. The simple analysis set-up also enables a rapid comparison of different non-linear regression curve fits and interpolation methods to quickly determine the best method. Consistent with Herman et al. (2008), these results demonstrate that polynomial methods for calibration curve fitting and interpolation are well-suited for the Lumit™ IL-1β (Mouse) Immunoassay.