Optimization of Capture ELISAs for Chicken Cytokines Using Commercially Available Antibodies

SIMPLE SUMMARY: Cytokines are small proteins that are involved in the communication between immune cells. Different cytokines have distinct functions and play thus an important role in orchestrating the response of the immune system. The detection of cytokines is generally based on either mRNA analysis or the detection of protein levels via an immunoassay. However, in poultry research, tools for cytokine detection at the level of proteins are not widely available. Therefore, our aim was to develop immune assays (capture ELISAs) to measure chicken cytokine proteins using commercially available antibodies and recombinant cytokine reagents. With our optimized protocols, we were able to detect individual cytokines in culture supernatants and serum or plasma in very low, physiologically relevant levels, in the developed capture ELISAs. These capture ELISAs can easily and inexpensively be utilized and may therefore have wide applicability in immunological research for poultry. ABSTRACT: Cytokines like interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-10, and IL-12p40 are important biomarkers for characterizing the nature and strength of immune responses. It is important to be able to quantify the cytokines at the protein level in biological samples. Quantification of chicken cytokines is generally performed on the level of messenger RNA (mRNA) by quantitative polymerase chain reaction (qPCR) because very few capture ELISAs for the quantification of chicken cytokine proteins are commercially available. Here, we describe the optimization and validation of capture ELISAs for chicken IL-2, IL-6, IL-10, IL-12p40, and IFN-γ using commercially available antibodies and reagents. First, we determined the optimal concentrations of the antibodies. We then verified the ELISAs’ performance and established that the lower limit of detection (LLOD) for all cytokines was below 32 pg/mL. The ELISAs show the same binding characteristics for recombinant and native cytokines (parallelism was <15.2% CV). Values for inter-assay variation were consistently low and mostly <20% CV. Overall, the optimized capture ELISAs are sensitive (<32 pg/mL) and reliable tools to quantify chicken cytokines. These ELISAs can easily and inexpensively be utilized in any immunological lab and may therefore have wide applicability in immunological research for poultry.