Endocrine Abstracts

Background: Excess adrenocorticotrophin (ACTH), most often from a pituitary tumour, results in the overproduction of cortisol and the condition, Cushing’s syndrome. Plasma ACTH measurements are used in the diagnosis of Cushing’s syndrome but due to the presence of multiple dibasic cleavage sites within its sequence, ACTH is extremely labile and far from an ideal analyte. 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 immunoassays. Furthermore, cross-reactivity with the precursor, pro-opiomelanocortin (POMC), increases the likelihood of erroneous interpretations and unreliable results.

Objectives: Here we explore the use of another POMC-derived peptide, the 30 amino acid (aa) joining peptide (JP), as a more robust surrogate measure of secreted ACTH levels. As it is not subject to further trypsin-like degradation, the JP is expected to have a much longer half-life than ACTH in plasma.

Methods: Antiserum was raised in sheep against synthetic 30 aa human POMC JP conjugated to keyhole-limpet hemocyanin (KLH). N- and C-terminal specific antibodies were affinity purified and subsequently used to develop ELISAs for the direct measurement of the dimeric endogenous JP in unextracted human plasma.

Results: The sensitivity of the assay was 10 ± 0.5 ng/l (n=8). The mean coefficient of variation was 6.8% within-assay and 5.5% between-assay and is less than 10% between 20 and >5000 ng/l. Initial experiments show endogenous JP immunoactivity is stable in vitro at 18-22°C for at least 24 hrs in plasma and serum. In comparison, the half-life for ACTH in plasma is <30 mins.ConclusionsThe assay is now being evaluated in normal subjects and patients with Cushing’s disease, ectopic Cushing’s syndrome and small-cell lung carcinomas. To conclude, the two-site ELISA for POMC JP in unextracted plasma could offer a reliable surrogate assay for clinical purposes.