Cd138 elisa kit

Cell-bound and shed forms of syndecan-1 were quantified using the CD138 ELISA Kit (Diaclone Research, Besancon, France) according to the instructions of the manufacturer. Conditioned, serum-free media were concentrated using Centricon centrifugal filter devices with 10 kDa nominal molecular weight limit filters (Merck Millipore, Burlington, MA, USA). Cells were lysed in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% v/v Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich). The protein content of the samples was determined by Coomassie reagent (Bio-Rad Laboratories Inc.) and 300 μg total protein from each sample was applied to the ELISA plate. Optical densities were read at 450 nm in a LabSystems Multiskan MS microplate reader (Thermo Fisher Scientific).

Maternal plasma soluble syndecan-1 concentrations were measured with a commercially available human syndecan-1 enzyme-linked immunosorbent assay (CD138 ELISA Kit, Diaclone), according to the manufacturers’ instructions. Plasma GAGs (heparan sulphate, dermatan sulphate and keratan sulphate) were measured in a subset of participants (that had sufficient material for analysis left) using high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) as previously described in detail^{36 (link),37 (link)}. heparan sulphate, dermatan sulphate, and keratan sulphate were first enzymatically digested into disaccharide units and then quantified on a Waters Quattro Premier XE (tandem) mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an Acquity UPLC system (UPLC-MS/MS). According to the nomenclature by Lawrence et al.^{37 (link)}, heparan sulphate was represented by the sum of disaccharides D0A0, D0S0, D0A6 and D2A0, D0S6 and D2S0, dermatan sulphate was represented by D0a4 and D0a10 and keratan sulphate was represented by the sum of disaccharides G0A6 and G6A6. All samples were analyzed in triplicate³⁴ .