Purification and quantitative measurement of bovine serum amyloid-A.
Bovine serum amyloid-A (b-SAA) was purified from a pool of acute phase serum using hydrophobic interaction chromatography and gel filtration. Serum was applied at a low salt concentration to a phenyl-sepharose column and SAA was eluted with a gradient of 0 to 6 M guanidine-HCl. Fractions containing SAA were pooled, concentrated and further purified by gel filtration on Superose-12. The concentration of SAA in bovine serum was quantified by an indirect ELISA using rabbit anti-human SAA and horseradish peroxidase conjugated donkey anti-rabbit IgG. Dilutions of an acute phase bovine serum sample were used as working standards. The SAA concentration of this standard was determined by comparison with purified b-SAA on SDS-polyacrylamide gel electrophoresis followed by densitometry at 590 nm. The assay detection limit was 3 micrograms ml-1; the intra-assay coefficient of variation was 4 per cent and interassay coefficients of variation were 5.5 per cent and 7.2 per cent at 66 and 178 micrograms ml-1 SAA, respectively. In calves experimentally infected with Pasteurella haemolytica type A1 the ELISA was able to detect a 10-fold increase of SAA within 24 hours of inoculation.
Horadagoda
A
A
Department of Veterinary Clinical Biochemistry, University of Glasgow, Bearsden.
Eckersall
P D
PD
Alsemgeest
S P
SP
Gibbs
H A
HA
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle / Q000097:blood*; D002845:Chromatography; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D006801:Humans; D015203:Reproducibility of Results; D000685:Serum Amyloid A Protein / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification
D000685:Serum Amyloid A Protein