Assay Linearity and Spike-Recovery Assessment in Optimization protocol for the analysis of Serum Cytokines by Sandwich ELISA Platform| Biomed Grid

Background: A sandwich Enzyme-linked immunosorbent assay (sELISA) can be used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as quality control checks in industries. To generate quality and reliable data using this technique, it is essential to optimise the conditions of carrying out sELISA. The optimisation protocol for sELISA includes but not limited to the determination of the right dilution of the sample type, the optimal concentration for the primary antibodies, the use of correct diluent to mention a few. Aim of study: This study provides a practical approach to establish the right working dilution of serum samples for cytokine sELISA. Study volunteers and methods: We carried out Linearity of Dilution (LD) assay to measure sELISA accuracy and reliability for interleukin (IL)- 4, IL-6, IL-8, IL-9, FGF-basic and granulocyte-macrophage-colony-stimulating factor (GM-CSF) using serially diluted serum samples from healthy volunteers and controlled by human group AB serum (hABs). Also, we undertook spike and recovery (SAR) assay to evaluate for interfering factors in the sample matrix. Results: Optimum sELISA signals were as follows: IL-4 = Neat, IL-6 = 1:4, IL-8 = Neat, IL-9 ≥ 1:100, FGF-basic = 1:8 and GM-CSF = Neat. The Spike & Recovery assay (SAR) data revealed higher per cent recovery rates for IL-8 and IL-9. Conversely, the remaining cytokines (IL-4, IL-6, FGF-basic and GM-CSF) showed low per cent recovery show the acceptable recovery rate of 80-120%. Our study showed that serum cytokines exhibit different signal strengths on the sELISA platform to varying dilutions of serum samples. Conclusion: It is therefore recommended that before embarking on serum cytokine sELISA analysis, multiple serial dilutions of serum samples must be used to determine the optimum working dilution for the respective cytokines to be adopted for the rest of the study