Milliplex map human circulating cancer biomarker magnetic bead panel 1

All analyses were performed in the laboratory of the coordinating center (Molecular Oncology Group, Medical University of Vienna, Vienna, Austria) employing the multiplex bead-based Luminex^®/xMAP^® technology. The 96-well plate assay panel (MILLIPLEX MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1; EMD Millipore, Billerica, MA, USA) was used to quantify the six proteins (CA125, HE4, OPN, PRL, leptin, and MIF) according to the manufacturer’s instructions. After completion of all incubation and washing steps the 96-well plate was read with the Bio-Plex™ 200 array reader (Bio-Rad Laboratories, Hercules, CA, USA). Raw data were analyzed with the Bio-plex Manager Software vs 6.1 (Bio-Rad). The Median Fluorescent Intensity (MFI) was assessed using a 5-parameter logistic curve-fitting method to calculate the concentration of all analytes.

Biomarker measurements were performed in duplicate on stored frozen plasma samples from the day of study enrollment. Plasma levels of IL-6, LBP, and IL-2R were measured using the IMMULITE immunoassay system (Siemens Medical Solutions Diagnostics, Germany). Additionally, plasma levels of ELA2, HGF, IL-6, IL-8, LTF, NGAL, resistin, sFas, TSP-1, and TRAIL were measured with the MILLIPLEX^® MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 and the MILLIPLEX^® MAP Human Sepsis Magnetic Bead Panel 3 (Merck Millipore, Germany) on a Luminex MAGPIX^® following the manufacturer’s instructions (Luminex Multiplexing Instrument, Merck Millipore). Biomarker levels below the lowest standard were extrapolated corresponding to the equation of the standard curve.

Antipsychotic-induced hyperprolactinemia is a proxy indicator of D₂ receptor occupancy manifesting when D₂ receptor occupancy is greater than 72% (96 (link)). Serum prolactin was assessed as a surrogate for central nervous system D2 receptor binding affinity for haloperidol and clozapine. Prolactin levels were determined from serum samples using the MILLIPLEX MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 (EMD Millipore, Burlington, MA, USA; Cat# HCCBP1MAG58K) containing prolactin coupled with the Luminex LX200 (EMD Millipore, Burlington, MA, USA) platform in a magnetic bead format according to manufacturer’s instructions. All serum samples were assayed in duplicate, and a pooled serum sample was included as a positive control. Concentrations are expressed as ng/ml. Data were analyzed using xPONENT v.3.1 software (EMD Millipore, Burlington, MA, USA). The minimum detectable concentration for prolactin was 30.2 pg/ml. The coefficient of determination for prolactin was 0.9999. The intra-assay precision was 4.9–15% and the inter-assay precision 4.1–16.2%. Intra-assay precision is generated from the mean of the %CV’s from 16 reportable results across two different concentrations of analytes in a single assay. Inter-assay precision is generated from the mean of the %CV’s across two different concentrations of analytes across 10 different assays.