Cytokine Biomarker Assays
Cytokines are promising biomarker candidates for predicting disease activity and response to therapy because of their prominent role in inflammation, immune response, and repair. However, the use of cytokines as biomarkers poses a few challenges, including a short serum half-life, very low (pg/mL) or undetectable serum baseline levels, potentially high disease- or drug-induced levels, lack of tissue- and toxicity-specific expression, and differential endogenous levels. Therefore, a highly sensitive cytokine biomarker assay is needed that can measure extremely low levels of proteins in biological matrices and has a large dynamic range.
A cytokine biomarker assay for the quantitation of interleukin-8 (IL-8) in cynomolgus monkey serum was developed and validated using the Gyrolab platform. Optimal parameters for quantitation were first determined, including capture and detection antibody concentrations, buffers, and matrix effects.
Recombinant IL-8 was then spiked into charcoal-stripped (protein-depleted) serum at predetermined levels and quantitated by immunoassay. The assay was validated by measuring the sensitivity, accuracy, precision, and selectivity of recombinant IL-8 in cynomolgus monkey serum (Figure 2).
The IL-8 immunoassay performed well over a wide range of analyte concentrations, ranging from 20 pg/mL to 10,000 pg/mL, and was selective for both recombinant and endogenous IL-8 as measured by spike, recovery, and parallelism, respectively. Drug treatment and disease state can result in the expression of cytokines at very high levels, and these samples must be diluted to the validated range of the assay.
To simulate theses conditions, high levels of IL-8 were spiked into serum and diluted to 1:2, 1:4, 1:8, 1:16, and 1:32. Nearly 100% recovery was observed for all dilutions, demonstrating dilutional linearity.
Finally, assay stability was measured. IL-8 remained stable over short-term (4 hr and 24 hr) stability testing in matrix at 15–30ºC and 2–8ºC; samples also remained stable (nearly almost 100% recovery) throughout six freeze-thaw cycles. Long-term stability will also be evaluated for up to one year. These data demonstrate that this cytokine biomarker assay is highly sensitive, can measure extremely low levels of proteins in biological matrices, and has a large dynamic range. In addition to IL-8, cytokine assays have recently been validated for IL-1β, IL-6, IL-10, IL-12, IFNγ, and TNFα.