Analysis by enzyme-linked immunosorbent assay and 2-dimensional electrophoresis of haptoglobin in the high-density lipoprotein fraction in cows

Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid me tabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipo protein (VHDL) fractions. The purpose of this study was to determine Hp con centrations in the lipoprotein fractions using an enzyme-linked immunosorbe nt assay (ELISA) based on the affinity with Hb, and also to detect struc tu ral differences of HDL Hp from that in the lipoprotein-deficient fraction u sing 2-dimensional electrophoresis. When purified Hp was used as the antige n for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtain ed from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA an d single radial immunodiffusion was 0.884. The ELISA was shown to be applic able to evaluate Hp concentrations in the lipoprotein fractions. Hp concent rations in the lipoprotein fractions were in the range of 0.94 to 8.77 mug of Hp/ml (n=4), and concentration ratios were 0.2 to 0.3% of whole serum Hp . Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VH DL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, alpha- and beta -chains of serum Hp were each separated into 5 spots, and their i soelectric point (pI) values were from 5.05 to 6.28 in the alpha -chain and from 5.92 to 6.95 in the beta -chain. The pi values of HDL Hp were indisti nguishable from those of serum Kp. These results indicate that the ELISA ba sed on the affinity with Hb is useful for evaluating Hp concentrations in l ipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction.