Blood samples were collected from dogs with AP (n = 22) and controls upon admission (n = 20), and also from dogs with AP following treatment (n = 9), at the time their clinical signs resolved. Blood was collected from the jugular or a peripheral vein, serum was separated from clotted whole blood by centrifugation at 1200 × g for 10 min, within 1 h of blood collection, and the serum was stored at −80 °C until assayed.
Serum CRP concentration was measured using a canine-specific ELISA kit (Canine C-Reactive Protein ELISA Kit, BD Biosciences, San Jose, USA), according to the manufacturer’s protocol; the intra-assay variability was <5%, the inter-assay variability was <10%, and the detection limit was 0.015 µg/mL. Because the amino acid sequence of HMGB1 is highly conserved among species, with 100% homology between humans and dogs (Murua Escobar et al. 2003 (link)), serum HMGB1 concentration was measured using a Human HMGB1 ELISA Kit (Human HMGB1 ELISA Kit, MyBiosource Inc., San Diego, USA), for which the intra- and inter-assay variabilities were <4.2% and <8.2%, respectively. The detection limit was 0.01 ng/mL. All samples, standards and controls were assayed in duplicate. The optical density was determined at 450 nm using an automated microplate reader (ELx 808, BioTek Instruments Inc., Winooski, USA).
Serum CRP concentration was measured using a canine-specific ELISA kit (Canine C-Reactive Protein ELISA Kit, BD Biosciences, San Jose, USA), according to the manufacturer’s protocol; the intra-assay variability was <5%, the inter-assay variability was <10%, and the detection limit was 0.015 µg/mL. Because the amino acid sequence of HMGB1 is highly conserved among species, with 100% homology between humans and dogs (Murua Escobar et al. 2003 (link)), serum HMGB1 concentration was measured using a Human HMGB1 ELISA Kit (Human HMGB1 ELISA Kit, MyBiosource Inc., San Diego, USA), for which the intra- and inter-assay variabilities were <4.2% and <8.2%, respectively. The detection limit was 0.01 ng/mL. All samples, standards and controls were assayed in duplicate. The optical density was determined at 450 nm using an automated microplate reader (ELx 808, BioTek Instruments Inc., Winooski, USA).