Optimization of a spectrophotometric method for quantification of acid-soluble glycoprotein in porcine serum.
The concentration of acid-soluble glycoprotein (ASG) in serum can be measured by selective precipitation with perchloric acid and then spectrophotometric quantification of the protein content in the supernatant. The purpose of this work was to standardize and optimize this procedure in pigs and to establish an automated protocol for quantification of the protein content in the supernatant with the use of bicinchoninic acid and copper sulfate. The greatest concentration of ASG was obtained when serum was incubated with 0.6 M perchloric acid at a 1:20 dilution. A minimum of 10 min of incubation at 37 degrees C or 25 degrees C was needed for complete selective precipitation. This method provided intra-assay coefficients of variation (CVs) of 5.14% and 5.12% and interassay CVs of 7.11% and 7.53% for samples with high and low ASG concentrations, respectively. The technique of linearity under dilution showed linear regression coefficients greater than 0.99. The limit of detection of ASG was 0.23 mg/mL, which is low enough to allow discrimination between normal and pathological states. A more than 2-fold increase in ASG concentration was found in pigs with postweaning multisystemic wasting syndrome when compared with healthy pigs. These results indicate that ASG could be considered a moderate acute-phase protein that would be easily measured and useful in assessing health status or the presence of inflammation in pigs. However, there was some overlap of results between groups of healthy and diseased animals.
Tecles
Fernando
F
Animal Medicine and Surgery Department, School of Veterinary Medicine, University of Murcia, Campus Espinardo s/n, 30100 Espinardo, Murcia, Spain.
Martínez-Subiela
Silvia
S
Cerón
José J
JJ
D000818:Animals; D015415:Biomarkers / Q000097:blood; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000032:analysis / Q000097:blood*; D010472:Perchlorates / Q000737:chemistry / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D013053:Spectrophotometry / Q000379:methods / Q000592:standards / Q000662:veterinary*; D013552:Swine / Q000097:blood*; D013553:Swine Diseases / Q000097:blood* / Q000175:diagnosis*; D013696:Temperature; D013997:Time Factors
D015415:Biomarkers; D006023:Glycoproteins; D010472:Perchlorates
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