Detectability of Cytokine and Chemokine using ELISA, following Sample-inactivation using Triton X-100 or Heat

Abstract

Background Routine characterization of clinical samples for their immunological responses against infectious pathogens often involves assessing cytokine/chemokine profiles and/or production of pathogen-specific antibodies. To prevent transmission of infectious materials in laboratories, these clinical samples are often inactivated by detergents or heat before the molecular assays are performed. Antibody-based molecular assays, such as ELISA, are highly sensitive to conformational changes in analytes. How specific inactivation procedures impact on cytokine/chemokine detectability in the clinical samples is not fully elucidated. This study compared two commonly used inactivation methods (Triton X-100, heat-inactivation) and untreated native samples in the cytokine/chemokine assays. Method Plasma, endotracheal tube aspirate (ETTA), and nasopharyngeal (NP) samples underwent inactivation with 0.05% Triton X-100 or heat (60°C, 1 hour). Cytokines/chemokine levels were assessed using Meso-Scale-Multi-Spot assays. Data were analyzed against untreated samples using one-way and Tukey’s multiple comparisons tests. Additionally, the conformational instability of cytokines/chemokines, predicted by their amino acid sequence, was examined to determine its contribution to detectability in inactivated samples. Results Heat treatment significantly impacted cytokine/chemokine detection across sample types. IL-1α levels were substantially reduced in ETTA, NP, and plasma samples. In heat-inactivated plasma, IL-12p40, IL-15, IL-16, VEGF, IL-7, and TNF-β, among 36 cytokines, were reduced by 33-99% (p-values ≤0.02). Conversely, Triton X-100 minimally affected cytokine/chemokine detection in plasma and NP samples by 11-37% (p-values ≤0.04). Triton X-100 increased the detection of IL-15, IL-16, IL-1α, VEGF, and IL-7 levels in NP samples. Triton X-100-inactivated ETTA samples showed no significant impact on cytokine/chemokine detectability. Heat inactivation had more profound impacts on protein detectability. Structural analysis revealed heat-affected cytokines had more hydrophobic residues and higher instability indices, although protein features alone could not reliably predict susceptibility. Conclusion Our findings demonstrated the importance of empirical assessments of inactivation protocols in the measurements of cytokine/chemokine responses in clinical samples. Overall, Triton X-100 performed better than heat inactivation in preserving protein conformation for antibodies-based immunological studies.