Preparation of anti-canine serum amyloid A (SAA) serum and purification of SAA from canine high-density lipoprotein.
Antiserum to canine serum amyloid A (SAA) was prepared in rabbits by immunization with crude SAA which was prepared from high-density lipoprotein 3 (HDL3) obtained from canine acute-phase serum. The antiserum was absorbed for contaminating antibodies by affinity chromatography using Sepharose 4B coupled with normal canine serum proteins. The rabbit anti-canine SAA serum reacted with a protein and formed a single precipitin line at the position of the alpha 1-region of the immunoelectrophoresis of canine acute-phase serum but did not react with the normal canine serum on immunoelectrophoresis. The antibody to canine SAA was also confirmed by Western blotting analysis. Canine SAA was purified as a low molecular weight protein component from crude SAA by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after gel filtration chromatography. Purified canine SAA had a molecular weight of 15,000 as estimated by SDS-PAGE. This SAA level was found by enzyme-linked immunosorbent assay (ELISA) to increase 1 day after inoculation with Bordetella bronchiseptica to 9.0-20.1 times the preinoculation value.
Yamamoto
S
S
Department of Immunology, Faculty of Environmental Health, School of Veterinary Medicine, Azabu University, Kanagawa, Japan.
Miyaji
S
S
Ashida
Y
Y
Otabe
K
K
Momotani
E
E
Rikihisa
Y
Y
D000210:Acute-Phase Reaction / Q000097:blood / Q000662:veterinary; D000818:Animals; D001885:Bordetella Infections / Q000097:blood / Q000662:veterinary; D016950:Bordetella bronchiseptica; D002846:Chromatography, Affinity / Q000662:veterinary; D002850:Chromatography, Gel / Q000662:veterinary; D004283:Dog Diseases / Q000097:blood; D004285:Dogs / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D007122:Immunoelectrophoresis / Q000662:veterinary; D008075:Lipoproteins, HDL / Q000737:chemistry*; D011817:Rabbits; D000685:Serum Amyloid A Protein / Q000302:isolation & purification*
D008075:Lipoproteins, HDL; D000685:Serum Amyloid A Protein