Development of a giant panda calprotectin-detecting immunoassay for non-invasive intestinal health monitoring

Abstract Non-invasive assessment of intestinal health in wildlife species remains a major challenge within One Health surveillance frameworks, particularly for protected or endangered animals. The giant panda (Ailuropoda melanoleuca), which exhibits unique digestive physiology and heightened sensitivity to dietary and environmental changes, lacks species-appropriate tools for monitoring intestinal inflammation. In this study, we developed an immunological framework based on recombinant giant panda calprotectin subunits. Recombinant giant panda S100A8 and S100A9 subunits were individually expressed using a Pichia pastoris system and used to generate monoclonal antibodies via hybridoma technology. Positive P. pastoris transformants were identified by colony PCR, and recombinant proteins were purified and verified prior to immunization. Following antibody screening and isotype characterization, systematic pairing assays were performed to identify compatible capture–detection antibody combinations suitable for sandwich ELISA development. The resulting monoclonal antibodies exhibited strong reactivity toward recombinant giant panda calprotectin, and multiple antibody pairs demonstrated antigen-dependent signals with low background reactivity. Although this study does not present a fully validated diagnostic assay, a semi-quantitative strategy for native calprotectin was established using immunoenrichment combined with SDS–PAGE and ImageJ-based densitometric analysis. Based on this approach, working standards were prepared, and a sandwich ELISA system was constructed. By enabling non-invasive monitoring of intestinal inflammation without reliance on cross-species reagents, this work contributes to the development of wildlife-appropriate health surveillance tools within a One Health framework and provides a reference approach for biomarker development in other non-model species.