Purification and quantitative measurement of bovine serum amyloid-A |
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Authors: | A. Horadagoda P.D. Eckersall S.P.M. Alsemgeest H.A. Gibbs |
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Affiliation: | a Department of Veterinary Clinical Biochemistry, University of Glasgow, Bearsden Road, Bearsden G61 1 QH, UK;b Department of Pathology, Faculty of Veterinary Medicine, University of Utrecht, PO Box 80185, 3508 TD Utrecht, The Netherlands;c Department of Veterinary Medicine, University of Glasgow, Bearsden Road, Bearsden G61 1 QH, The Netherland |
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Abstract: | 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-HCI. 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 μg 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 μg 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. |
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