Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood

María D. Parra Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, 30100 Espinardo campus, Murcia, Spain.

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Mika Tuomola Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland.
Present address is Raisio Life Sciences, PO Box 101, FIN-21201 Raisio, Finland.

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Juan Cabezas-Herrera Laboratory of Molecular Biology, Clinical Analysis Unit, University Hospital Virgin de la Arrixaca, El Palmar, Murcia, Spain, 30120.

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José J. Cerón Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, 30100 Espinardo campus, Murcia, Spain.

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Abstract

Objective—To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood.

Animals—12 healthy dogs and 35 dogs with inflammatory processes.

Procedure—CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4°C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases.

Results—Analytic and functional limits of detection were 0.53 and 3.26 µg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs.

Conclusions and Clinical Relevance—Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs. ( Am J Vet Res 2005;66:62–66)

Abstract

Objective—To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood.

Animals—12 healthy dogs and 35 dogs with inflammatory processes.

Procedure—CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4°C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases.

Results—Analytic and functional limits of detection were 0.53 and 3.26 µg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs.

Conclusions and Clinical Relevance—Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs. ( Am J Vet Res 2005;66:62–66)

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