Patients with primary SI-NETs were collected after surgery with a curative intent. Serum and plasma samples were obtained and analyzed from 66 lung carcinoid patients: 52 TC (named TLC) and 14 AC (named ALC). The median ages of the patients were SI-NET 63 years (range, 27�C82) and TLC & ALC 59 years (range, 13�C81). Moreover, 50 serum samples from healthy volunteers were collected at the Uppsala contain University Hospital and used as negative controls. Clinicians at the Uppsala University Hospital, Neuroendocrine Center of Excellence, Uppsala, Sweden and at the European Institute of Oncology, Milan, Italy that follow the SI-NET and lung carcinoid patients have never identified PNS and neurological symptoms.
Reagents and antibodies Maxisorp strips (NunC, Roskilde, Denmark), GST-PNMA2 recombinant protein (Abnova, Taipei, Taiwan); 3,3��,5,5-tetramethylbenzidine (TMB) + substrate (Dako, Glostrup, Denmark), Peroxidase with dakocytomation peroxidase block (Dako), Dakocytomation envision? system labeled polymer-HRP anti-rabbit kit and 3-3��-diaminobenzidine (Dako), Mayer’s hematoxylin (Histolab Product AB, Gothenburg, Sweden), Graded alcohol (Kemetyl, Vestby, Norway), Xylen (Solveco, Rosersberg, Sweden), Pertex? (Histolab, Gothenburg, Sweden), Tris-Glycine blotting buffer (Amresco, Solon, OH), Western blotting (WB) reagent and Lumi-Light WB substrate (Roche, Basel, Switzerland), EasyTag Methionine-L-35S, NEG709A005MC (PerkinElmer, Waltham, MA), Full-length cDNA clone for human PNMA2, ID6580976 (BioScience Geneservice, Cambridge, UK), TnT? SP6 Quick Coupled Transcription/Translation System (Promega, Madison, WI), Protein G-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK), Peroxidase (HRP)-conjugated rabbit anti human IgG (anti-IgG) (Dako), rabbit polyclonal antibody anti-PNMA2 (Atlas Antibodies, Stockholm, Sweden), monoclonal mouse anti-GST, sc-138, polyclonal goat anti-human Ma2, sc-68099, HRP-donkey anti-goat, sc-2020, (Santa Cruz Biotechnology, Santa Cruz, CA), HRP-goat anti-rabbit, P0448, (Dako) and Ravo PNS-Blot, (Ravo Diagnostika GmbH, Freiburg, Germany).
Indirect ELISA We set up a novel indirect ELISA to detect Ma2 autoantibody levels in sera and plasma of NET patients and healthy volunteers. The standard curves to screen both serum and plasma samples were constructed by serial dilutions 1100, 1200, 1400, 1800, 11,600, 13,200 and 16,400 using the serum and the plasma from the same patient with primary SI-NET, expressing anti-Ma2 used as reference.
Two controls were used to evaluate individual runs. One was from a patient with primary SI-NET, expressing high titer of anti-Ma2 and the other one was from a healthy donor expressing low titer of anti-Ma2. GST-PNMA2 recombinant protein (Abnova, Taiwan), diluted in the coating buffer Dacomitinib (15 mM Na2CO3, 35 mM NaHCO3, pH 9.