Validation of a multiplex assay for measurement of IL-6, IL-8, and TNF-alpha in sputum

<b>Introduction:</b> The pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-alpha (TNF-α), are strongly associated with the ‘cytokine storm’, an overreactive immune response that often progresses to systemic sepsis and death. Unfortunately, this biological complication is often observed in severe Covid-19 disease, with elevated plasma levels of IL-6, IL-8, and TNF-α a defining feature. However, as Covid-19 targets the pulmonary epithelium, a multiplex assay, that measures key cytokines in sputum, may assist in patient stratification. <b>Aims and Objectives:</b> To validate a multiplex assay for IL-6, IL-8 and TNF-α quantification in sputum. <b>Methods:</b> Sputum sol samples (n=40) were used in a range of validation experiments for each cytokine. Firstly, the degree of sputum sample matrix interference, during IL-6, IL-8, and TNF-α measurement, was examined; followed by an evaluation of assay precision, cross-reactivity, selectivity, and linearity. <b>Results:</b> Matrix interference was found to vary between sputum samples when measuring for each cytokine; and thus, samples were assayed at dilutions previously shown to abrogate this ‘matrix effect’. Intra and Inter-assay CVs were calculated as &lt;4 % and &lt;12%, respectively, for all three cytokines. Linearity and selectivity percentages all fell within the desired range of 80-120%, and a high degree of specificity was noted for each cytokine when tested against closely related molecules. <b>Conclusion:</b> This validation study highlights the suitability of this multiplex assay for IL-6, IL-8, and TNF-α quantification in sputum sol.