Capillary Zone Electrophoresis Procedure for Bovine Haptoglobin Determination by Measuring the Haemoglobin Binding Capacity: Precision and Sensitivity in Respect of Capillary and Canine Haemoglobin Characteristics

Andrea Alvarez, Marie-France Humblet, Lisette Trzpiot, Jean-Marie Godeau University of Liège, Faculty of Veterinary Medicine, Department of Functional Sciences, Biochemistry Unit, 20 Bvd de Colonster (B42), 4000 LIÈGE, Belgium Poster Presented At The Second European Colloquium on Acute Phase Proteins.University of Bonn, Germany,12 May 2001 Measuring the haemoglobin (Hb) binding capacity (HbBC) of haptoglobin for quantitative bovine haptoglobin (bHp) determination can be achieved by two ways. Skinner et al. in 1991 (Vet. Rec. 128, 147-149) described the first for bovine serum by recording at 510 nm the peroxidase activity of bovine haemoglobin (bHb) bound to Hp at pH 4.0 (classic photometric method; sensitivity: 10 mg/L). The second, by a capillary electrophoretic separation (CZE) of the complex HboHp from Hb free in the presence of canine Hb (cHb) added in excess to the serum. Quantitative bHp determination was achieved by recording the peak area of HboHp at 410 nm (Pirlot et al., 1999, 128, 147-149; capillary 25 m diam.). Nevertheless, the authors did not communicate any information on the sensitivity of CZE procedure. The lowest Hp solution used contained 220 mg/L, and the peak area recorded with such Hp preparation was very low. This suggests that CZE did not allow Hp determination below 150 or 100 mg/L in these conditions. The purpose of this work was an evaluation of CZE capacity for bHp determination around its cut-off point value as determined by our laboratory in cattle (20-40 mg/L). In order to improve the sensitivity, the first way could be the measurement of free cHb amount disappearance due to the presence of bHp, and the second an improvement of CZE conditions for lower cHbobHp amount determination. The use of fresh cHb or Cyanmethaemoglobin and Hb batch kept at 4oC were compared along time at different concentrations and in the presence of azide (NaN3, 3.7 mM) to prevent bacterial growth. Precision and sensitivity were determined for free Hb and HboHp complexes by using different capillaries (24 or 44 cm long; 25, 50 or 75 m diam). Most satisfactory conditions were finally defined for the lowest Hp concentration assayed, i.e. a solution of 29 mg/L (CV: 15-20%). The Federal Ministry of Agriculture supported this work. 1Andrea Alvarez is a fellowship from the ALFA VET network supported by the European Community.