SUN-251 Highly Sensitive ELISA System Enables Direct Detection of C-Type Natriuretic Peptide-53 in Plasma Without Extraction

Abstract Disclosure: D. Kosugi: None. N. Minamino: None. D. Taura: None. M. Makoto: None. M. Ohta: None. K. Nakao: None. Background and aim: Mammalian natriuretic peptide (NP) family is composed of A-type (Atrial), B-type (Brain) and C-type NP; ANP, BNP, and CNP. CNP is widely distributed in the central and peripheral tissues. CNP induces endochondral ossification, vasorelaxation, antifibrosis, and so forth as a local regulator, and its analog, vosoritide, is used for the therapy for achondroplasia. However, the clinical significance of CNP has not yet been established, mainly due to lack of methods sensitive enough for measuring plasma CNP without extraction. Although plasma CNP levels in normal subjects are presumed to be at 0.1-0.5 pM, many researchers have reported far higher CNP levels. In the present study, we have undertaken development of sandwich chemiluminescent ELISA (CLEIA) for CNP-53, the major circulating molecular form of CNP in plasma. Methods: Monoclonal antibodies targeting the ring portion and N-terminal region of CNP-53 were generated by immunizing rats or mice with respective peptide-KLH conjugates, followed by cloning and selection. Antibody #26-7 recognizing the N-terminal region was biotinylated, fixed on the avidin plate, and used for capture antibody. Antibody #47-22 recognizing the ring portion was used for the detection antibody after labeling with alkaline phosphatase, where CDP-star/Emerald II was used as a substrate. Immunoreactive(ir) CNP-53 concentrations in plasma from healthy humans and Wistar Kyoto rats were measured. Results: The detection and quantitation limit of the developed CLEIA was 0.015pM and 0.041pM, respectively, and CNP-53 was measurable up to 250pM. This CLEIA showed equivalent recognition of mammalian CNP-53, including human and rat CNP-53, without cross-reactivity to CNP-22, ANP, BNP, or other related bioactive peptides, except for equal recognition of proCNP and CNP-53. In this CLEIA, plasma samples were measured after four-fold dilution and standard curves were generated with peptide-free-plasma. Plasma levels of irCNP-53 in healthy human subjects were 0.18±0.06 pM, and those in Wistar Kyoto rats were 0.24±0.02 pM. Further studies on plasma irCNP-53 from disease model rats and patients are ongoing in our laboratory. Conclusion: The newly developed CLEIA for CNP-53 demonstrated superior sensitivity and quantitation capabilities, allowing for direct and highly accurate measurement of plasma irCNP-53 concentrations. This method provides a more reliable and reproducible approach than previously reported assays. Utilizing this CLEIA, more detailed physiological and pathophysiological changes of plasma CNP will be elucidated in near future. Presentation: Sunday, July 13, 2025