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Analytical and Clinical Validation of a Time-resolved Immunofluorometric Assay (TR-IFMA) for Canine C-reactive Protein in Serum

Veterinary Research Communications volume 30, pages 113–126 (2006)Cite this article

Abstract

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of C-reactive protein (CRP) in canine serum. CRP was isolated from canine acute-phase serum by affinity chromatography on agarose coupled with phosphorylethanolamine. This isolated dog CRP was used as standard to calibrate the assay. Intra-assay and inter-assay coefficients of variation were in the ranges 5.3–7.1% and 4.8–13.3%, respectively. Accuracy, evaluated by adding 2 and 10 μg/ml of CRP to serum samples, provided recoveries of 99.9% and 106.8%. High correlation was found between CRP measurements by TR-IFMA and a by commercial enzyme-linked immunosorbent assay (R 2 = 0.98). The limit of detection for the TR-IFMA method was 0.000067 μg/ml and the measurement of CRP in serial dilutions of acute-phase dog sera generated curves with the same slope as the one constructed with purified CRP. The TR-IFMA provides a precise, accurate and highly sensitive assay for CRP determination in dog samples. CRP levels in dogs with different diseases ranged between 10.2 and 210.7 μg/ml and were significantly higher than those observed in healthy dogs (< 7.1 μmg/ml).

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Abbreviations

BITC:

biotin isothiocyanate

BSA:

bovine serum albumin

CRP:

C-reactive protein

CV:

coefficient of variation

ELISA:

enzyme-linked immunosorbentassay

PEAE-agarose:

agarose coupled with phosphorylethanolamine through epoxy bond

SD:

standard deviation

SDS-PAGE:

sodium dodecyl sulphate–polyacrylamide gel eletrophoresis

TR-IFMA:

time-resolved immunofluorometric assay

TSA:

Tris-HCl containing 150 mmol/L NaCl and 0.02%NaN3

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Authors and Affiliations

  1. Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, 30100, Murcia, Spain

    M. D. Parra & J. J. Cerón

  2. Department of Biotechnology, University of Turku, Turku, Finland

    M. Tuomola

  3. Laboratory of Molecular Biology, Clinical Analysis Unit, University Hospital Virgen de la Arrixaca, El Palmar, Murcia, Spain

    J. Cabezas-Herrera

Authors
  1. M. D. Parra
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  2. M. Tuomola
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  3. J. Cabezas-Herrera
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  4. J. J. Cerón
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Correspondence to M. D. Parra.

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Parra, M.D., Tuomola, M., Cabezas-Herrera, J. et al. Analytical and Clinical Validation of a Time-resolved Immunofluorometric Assay (TR-IFMA) for Canine C-reactive Protein in Serum. Vet Res Commun 30, 113–126 (2006). https://doi.org/10.1007/s11259-006-3201-z

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  • DOI: https://doi.org/10.1007/s11259-006-3201-z

Keywords

  • C-reactive protein
  • dog
  • serum
  • time-resolved immunofluorometric assay
  • validation