Intestinal transplantation (ITx) is the definitive treatment for intestinal failure (Sudan et al., 2000; Kaufman et al., 2020). However, ITx intervention is limited by high rates of rejection and graft-versus-host disease (GVHD) compared to that of other solid organ transplants. (Kaufman et al., 2020; Mayer, 2000; Zhang et al., 1996; Ganoza et al., 2019) These complications necessitate aggressive immunosuppression, which carry the risk of adverse events, such as posttransplant lymphoproliferative disorder and infections (Shpilberg et al., 1999; Roberts and Fishman, 2021; Sudan, 2014). Despite the profound consequences of rejection, current methods of early diagnosis where intervention might be possible have shortcomings, including endoscopic biopsy, the current gold standard for diagnosing rejection. Patchy distribution of rejection may require multiple invasive biopsies for accurate histopathology (Wu et al., 2003), and the subjective nature of biopsy interpretations, such as “borderline rejection”, can result in indecisive management (Malone, 2021). As rejection episodes can occur many times over the course of multiple years post-transplant, there is a need for reliable, resource-light methods for the diagnosis and treatment of rejection after ITx.

Antimicrobial peptides (AMPs) help mediate mucosal homeostasis in the intestine with luminal microbes. Changes in AMP levels within secretory granules of intestinal Paneth cells have been associated with intestinal graft rejection as well as Crohn's disease (Fishbein et al., 2008a; Clevers and Bevins, 2013). We hypothesize that levels of AMPs in the ileal effluent might associate with rejection; if so, they could be serially monitored from stoma non-invasively and serve as biomarkers for early ITx rejection, which otherwise requires histological analysis of biopsy specimens (Sung et al., 2016). To date, measurement of AMPs in ileal fluid through stoma sampling has not been reported. Of interest would be analysis of myeloperoxidase, calprotectin, β-defensin 2 (HBD2, DEFB2) and lysozyme; ELISA kits for these targets are commercially available but have not been adapted for use in ileal effluent. Another attractive target is α-defensin 5 (HD5, DEFA5), an abundant Paneth cell-specific effector (Mallow et al., 1996). Commercial ELISA kits are not available for HD5. Creating an HD5-specific ELISA would permit correlation of prior findings of mucosal HD5 to luminal HD5. Together, assessment of levels in ileal fluid through stoma sampling of these five AMPS might provide a noninvasive method of assessing mucosal innate immune barrier function, Paneth cell function, and indirectly, dysbiosis. In the present study, we describe the development and optimization of assays to detect AMPs in ileal stomal effluent, including the development of a sandwich ELISA assay for HD5.