Development of a highly sensitive immunoassay for the detection of peripherin in blood

Introduction: Peripherin (PRPH), as a member of the neurofilament family, is specific to the peripheral nervous system and might therefore be a suitable biomarker in monitoring and diagnosing peripheral neuropathies, such as Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP). Furthermore, PRPH could also potentially aid in distinguishing peripheral neuropathies from central nervous system disorders. However, analyzing PRPH in fluid samples remains challenging due to its low levels and blood matrix interferance. The aim of this study was to establish a highly sensitive immunoassay to reliably measure and quantify PRPH in blood. Methods: Three highly sensitive immunoassay platforms – the Ella, Mesoscale Discovery (MSD) and Simoa platforms – were employed to develop sandwich immunoassays for PRPH detection in serum. Optimization was performed on all three platforms, but subsequent technical and clinical validation was only performed on the Simoa due to limitations on the Ella and MSD platforms. Key optimization steps included adjusting the incubation time, applying mechanical force, and adding chemicals to the buffers to enhance the signals. Finally, the established assay was used to analyze serum samples from patients with GBS (n = 8), CIDP (n = 10), Alzheimer’s disease (AD) (n = 9), amyloid lateral sclerosis (ALS) (n = 9) and controls (n = 7). Results: Initial antibody testing was performed on both the Ella and MSD platforms. However, both platforms faced limitations due to sensitivity and stability issues, respectively. Subsequently, the assay was established on the ultrasensitive Simoa platform. The final developed PRPH assay displayed intra- and inter-assay coefficient of variations below 20%. The established assay was highly sensitive, with a lower limit of quantification and detection of 3.05 pg/ml and 0.92 pg/ml, respectively. Serum PRPH levels measured in the five diagnostic groups showed no significant differences between all groups with a tendency towards higher PRPH levels in CIDP and GBS patients. However, CIDP PRPH levels were significantly higher than those in the control and AD groups when compared in a two-group analysis (both p < 0.05). Conclusion: A highly sensitive immunoassay could be developed for the analysis of PRPH in blood samples. The assay demonstrated good technical validation and increased PRPH concentrations in peripheral neuropathies. The assay’s high sensitivity allows reliable quantification of low PRPH levels, facilitating clinical research applications. In future experiments larger clinical cohorts need to be assed to reliably assess the clinical applicability of the novel PRPH assay