Experimental evaluation of western blotting for serum cytokine analysis

Interleukin-9, or IL-9, plays a role in allergic inflammation, autoimmunity, and tumor immunity. Despite its biological relevance, measuring IL-9 in human serum is hard. The levels are usually low, and most standard protein detection techniques are not sensitive enough to detect it. This study aimed to determine the detection threshold of IL-9 using chemiluminescent Western blotting and evaluate its applicability for measuring endogenous IL-9 levels in serum samples from healthy individuals. We performed serial dilutions of recombinant IL-9 from 20 ng to 0.01 ng and analyzed them alongside serum samples (S15, S10, S5) from healthy volunteers by Western blotting. Signal intensity for each was assessed by subtracting the background signal to determine the detection limits. Membrane stripping, three-layer antibody amplification, immunoprecipitation, and prolonged exposure were trialed to enhance sensitivity as part of an optimization approach. IL-9 was effectively detected at concentrations ≥5 ng, with signal intensity dropping nattily below this threshold. Serum samples showed a negligible signal, implying that endogenous IL-9 concentrations in healthy individuals fell below the assay’s detection limit. Also, signal enhancement strategies failed to improve detection at lower concentrations. Western blotting is suitable for confirming the presence of IL-9 at nanogram levels but is insufficient for detecting physiological concentrations in serum. These findings underscore the need for more sensitive platforms, such as ELISA or digital immunoassays, to accurately quantify low-abundance cytokines, such as IL-9, in clinical samples.