Determination of Serum Haptoglobin Levels by Cellulose Acetate Electrophoresis

Basic conditions for the application of cellulose acetate electrophoresis to the quantitation of serum haptoglobin levels were investigated and a convenient method was established. This method consisted of addition of hemoglobin into serum to a final concentration of 300mg/dl, and applying a 4μl aliquot of the serum on a strip of cellulose acetate mambrane at 1/3 from the cathode end. Electrophoresis was carried out at 0.5mA/cm and at 110-140V for 1 hour in a 0.05M phosphate buffer, pH 7.0. After drying the membrane, it was stained with o-dianisidine for 10 minutes and washed with distilled water for an additional 10-minute period. Densitometric scanning was carried out at 430mμ. When the concentration of added hemoglobin was less than 300mg/dl, the densitometric reading of free hemoglobin was found to be almost always 1.25 times that of haptoglobin-hemoglobin complex.It was, therefore, necessary to correct densitometric data by the following equation in order to get haptoglobin levels;Hp(mg/dl)=300×1.25(Hp-Hb)/methemalb.+1.25(Hp-Hb)+free Hb