Plasma adrenocorticotrophin (ACTH) is extremely labile and far from an ideal analyte for use in the diagnosis of Cushings syndrome. Processing of ACTH by some ectopic tumours releases high levels of smaller ACTH-like fragments, α-MSH and CLIP, which can interfere with individual antibodies in current diagnostic immunoassays. Furthermore, cross-reactivity with the precursor of ACTH, pro-opiomelanocortin (POMC), increases the likelihood of erroneous interpretations and unreliable results. Here we explore the use of other co-secreted POMC-derived peptides, pro-y-MSH and the POMC joining peptide (JP), as more robust surrogate measures of secreted ACTH levels. For detection of pro-y-MSH, a two-site immunoassay was constructed using antiserum raised in rabbits and sheep against N-POMC (1-28) and y1-MSH, respectively. For detection of the POMC JP, antiserum was raised in sheep against the full-length peptide, and N- and C-terminal-specific antibodies were affinity purified and subsequently used to develop a two-site immunoassay. Both ELISAs were optimised for the direct measurement of endogenous pro-y-MSH and POMC JP in unextracted human plasma. The sensitivity of the pro-y-MSH assay was 12 ± 0.3 ng/l (n=10) and the JP assay was 10 ± 0.5 ng/l (n=8). Initial experiments show that endogenous pro-y-MSH and JP remain stable in vitro at room temperature for far longer than ACTH, which has a half-life in plasma of <30 minutes. The analytical performance of the assays will be assessed, along with a comparison of endogenous pro-y-MSH and JP levels in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The assays are now being evaluated in patients with Cushings disease, ectopic Cushings syndrome and small-cell lung carcinomas. To conclude, the two-site ELISAs for pro-y-MSH and POMC JP in unextracted plasma could offer reliable surrogate assays for clinical purposes.
14 Nov 2022 - 16 Nov 2022