Sigmatrix serum diluent

Primary experiments were performed in assay buffer and artificial matrices (SigMatrix (Serum Diluent, Merck KGaA, Darmstadt, Germany); SeraSubTM (CST Technologies, Inc., New York City, NY, USA)). Diluent 2 (Meso Scale Discovery, LLC., Rockville, MD, USA) for PD-1 and PD-L2 and PBS with 1% BSA for PD-L1 were used. In the second set of experiments, heparin plasma from up to eight donors was used. Probes were used native or spiked to a concentration of at least 10 ng/mL with recombinant protein from R&D Systems (R&D Systems, Inc., Minneapolis, MN, USA). After that, a serial 1:1 dilution row was prepared including dilutions 1:2; 1:4; 1:8 and, if applicable, 1:16 in the respective assay buffer or plasma.

Analytical standards (purity > 98%) of GnRH and l-LHRH-III were purchased from GeneCust (Chalmont, France). Stock solutions were prepared with a concentration of 1000 ng/mL using methanol and were stored at − 20 °C until use. Further dilutions were obtained in 0.1% formic acid in water using plastic vials in order to avoid adsorption. All aqueous solutions were prepared with HPLC-grade water from a Milli-QAcademic System (Millipore, Burlington, MA, USA). Acetonitrile hyper-grade for LC–MS, formic acid, and TFA (trifluoroacetic acid) were purchased from VWR International (Radnor, PA, USA); acetic acid and SigMatrix Serum Diluent were purchased from Merck (Darmstadt, Germany). Human plasma samples were provided by “Centro Produzione e Validazione Emocomponenti (C.P.V.E.) SC Banca del Sangue, Città della Salute e della Scienza” Torino, Italy. The fresh human plasma refrigerated was maintained at the temperature of + 4 °C until use. We employed ten different lots of human plasma refrigerated, previously analyzed to detect traces of GnRH. Each lot was used for the validation protocol only in the case of undetectable GnRH (< LLOQ, vedi infra).