Hrp conjugated donkey anti mouse igg

Mouse blood was collected from the inferior vena cava at the end of the PV-loop procedure. After incubation for 1–2 h at room temperature, the serum was prepared by centrifugation at 300 ×g followed by centrifugation at 16 000 ×g for 20 min, aliquoted and kept frozen at −80 °C. Antibodies to β2GPI were measured by ELISA using 96-well plates coated with human β2GPI supplied with the β2GPI-IgG ELISA kit (Inova Diagnostics, San Diego, CA, USA). Serum samples were diluted 1 : 50 with the sample diluent from the kit, incubated for 30 min on a plate, probed with HRP-conjugated donkey anti-mouse IgG (ab7061; Abcam, Cambridge, MA, USA) and detected with tetramethylbenzidine (TMB) substrate. Each set of measurements contained serial dilutions of mouse monoclonal anti-human β2GPI IgG (Alpha Diagnostic, San Antonio, TX, USA) prepared in the sample diluent and used to generate a standard curve. Mouse anti-dsDNA total Ig (Alpha Diagnostic) and Cystatin C (R&D Systems, Minneapolis, MN, USA) ELISA measurements were performed according to the manufacturer’s instructions. The blood urea nitrogen (BUN) colorimetric detection kit used was from B-Bridge International (Santa Clara, CA, USA). Total cholesterol was measured with the Amplex Red cholesterol assay kit (Invitrogen, Carlsbad, CA, USA) and the triglyceride quantification kit was from Abcam.