ECE2020 Audio ePoster Presentations General Endocrinology (17 abstracts)
Vasoinhibin dimerization and measurement in human sera with a new vasoinhibin elisa
Jakob Triebel 1 , Nils Müller 1 , Johannes Ebnet 1 , Leon Neugebauer 1 , David Harris 1 , Christin Friedrich 1 , Hülya Markl-Hahn 1 , Juan Pablo Robles 2 , Magdalena Zamora 2 , Gonzalo Martínez de la Escalera 2 , Carmen Clapp 2 & Thomas Bertsch 1
1Institute for Clinical Chemistry, Laboratory Medicine and Transfusion Medicine, General Hospital Nuremberg & Paracelsus Medical University, Nürnberg, Germany; 2Instituto de Neurobiología, Universidad Nacional Autónoma de México (UNAM), Mexico
Vasoinhibin, historically also known as 16K PRL, is a peptide hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by proteolytic cleavage of prolactin (PRL). The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the development of new pharmacological treatments and their evaluation in clinical interventional trials (ClinicalTrials.gov Identifier: NCT03161652 and NCT00998556). Recent insights into the function and three-dimensional structure of vasoinhibin revealed a structure conformed by a three-helix bundle and some degree of dimerization. To study the level of vasoinhibin dimerization and the presence of vasoinhibin in human serum, recombinant human vasoinhibin and human sera were investigated by SDS-PAGE and Western blotting, preparative electrophoresis, mass spectrometry, immunoprecipitation, and ELISA. Commercial poly- and monoclonal anti-PRL antibodies were used, and monoclonal anti-vasoinhibin antibodies, designed to specifically bind vasoinhibin and not PRL, were generated by the hybridoma technique. A sandwich ELISA for vasoinhibin was developed, using anti-vasoinhibin monoclonal antibodies. Over 90% of recombinant vasoinhibin monomers aggregated to form a 28 kDa dimer which is resistant to standard denaturating and reducing conditions and dissociated only after prolonged exposure to heat and ß-mercaptoethanol or DTT. Western blotting analysis and immunoprecipitation demonstrated the presence of a highly abundant, 28 kDa endogenous protein in human sera, with immunochemical features of vasoinhibin. The ELISA, calibrated with a recombinant human vasoinhibin-standard, had a detection limit of 39 ng/ml, a quantitation limit of 207 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. Serum samples from 9 individuals were tested, and showed variable concentrations ranging from 1 to 210 µg/ml. The contribution of endogenous vasoinhibin to these measurements is under investigation.
Volume 70
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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