Characterization of Haptoglobin from the Serum of Captive Bottlenose Dolphins (Tursiops truncatus) and its Application to the Monitoring of Acute Phase Response
Haptoglobin (Hp) is known to be an acute phase protein and has been reported to increase in serum from Stellar sea lions and harbor seals during inflammation, infection, trauma and tumor. The purpose of the present study was to characterize the serum Hp in the dolphins (dlHp) and to utilize the dlHp antibody for monitoring acute-phase inflammatory response in these animals.
Purification of serum dlHp was performed by precipitation using ammonium sulfate (33 percent and 60 percent) and antibody-affinity chromatography. Purified dlHp was identified with immunoblot analysis and N-terminal amino acid sequence. Relative concentration of serum dlHp was determined by immunoblot analysis and NIH image.
Serum dlHp was detected by immunoblot analysis using anti-canine Hp IgG, and was not detected with anti-bovine Hp IgG. The fraction containing dlHp, apo-A1 and IgG was obtained by antibody-affinity chromatography with anti-canine Hp IgG after precipitation by 60 percent saturated ammonium sulfate. After delipidation using n-butanol and diisopropyl ether, the extract was subjected to the antibody-affinity chromatography, and the fraction containing dlHp was obtained. DlHp was purified by elution electrophoresis. The purified protein had a molecular mass of 99.3 kDa and contained 36.2 kDa peptides linked by disulfide bonds. N-terminal amino acid sequence analysis revealed that these peptides were homologous to rabbit Hp, rat Hp and dog Hp. Serum dlHp was detected using anti-dlHp IgG prepared in this study. Serum dlHp levels from dolphins with elevated leukocyte counts were significantly higher than those from control dolphins. DlHp was detected in polymorphonuclear leukocytes of the dolphins using immunoblot and immunohistochemical analyses.
Serum dlHp was purified by antibody-affinity chromatography using anti-canine Hp IgG. Increasing serum dlHp levels in dolphins with elevated leukocyte counts suggest that serum dlHp is one of the useful markers for monitoring acute phase response in captive bottlenose dolphins.