Quantitative determination of haptoglobin (HAP) in human and bovine sera by capillary zone electrophoresis (CZE).
This study describes an original assay for serum haptoglobin determination by measuring the capacity of human haptoglobin (hHAP) and bovine haptoglobin (bHAP) to bind haemoglobin (Hb) as established by capillary zone electrophoresis (CZE). This method involves the addition of Hb in excess to the serum and the separation of the HAP-Hb complexes from free haemoglobin by CZE. Protein migration was recorded at a wavelength of 415 nm which reveals Hb alone (free or bound), and the concentration of HAP was indirectly estimated by measuring bound Hb. Different CZE conditions and the peak migration time of Hb from various species (human, equine, bovine, canine) were investigated. The electrophoretic separation of free human Hb (hHb) in excess and the hHAP-hHb complex was fully achieved by CZE, allowing a quantitative determination of hHAP. However, bovine haemoglobin (bHb) bound to bHAP and free bHb were poorly separated under the same conditions. The best detachment between bHAP-Hb complexes and free Hb was only attained in the bovine sample by use of canine haemoglobin (cHb). CZE assays performed with cHb gave very close values to those of a classic photometric method which measured the peroxidase activity of the haptoglobin-cyanmethaemoglobin complexes (y = 1.0168x - 0.072; r2 = 0.97). CZE assay was fast (< 10 min), inexpensive, did not require the use of a specific antibody and was reproducible (coefficient of variation, CV 3.6%).
Pirlot
A
A
Département de biochimie générale et biochimie clinique, faculté de médecine vétérinaire, Université de Liège, Belgium. A.Pirlot@ulg.ac.be
Janssens
J
J
Skinner
G
G
Godeau
J M
JM
D000818:Animals; D002417:Cattle; D004285:Dogs; D019075:Electrophoresis, Capillary / Q000379:methods; D006242:Haptoglobins / Q000032:analysis* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods
D006242:Haptoglobins; D006454:Hemoglobins