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

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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

References

  • Adlercreutz, H., Wang, G.J., Lapcik, O., Hampl, R., Wähälä, K., Mäkelä, T., Lusa, K., Talme, M. and Mikola, H., 1998. Time-resolved fluoroimmunoassay for plasma enterolactone. Analytical Biochemistry, 265, 208–215

    Article  PubMed  CAS  Google Scholar 

  • Aggerbeck, H., Norgaard-Pedersen, B. and Heron, I., 1996. Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay. Journal of Immunological Methods, 190, 171–183

    Article  PubMed  CAS  Google Scholar 

  • Caspi, D., Baltz Marilyn, L., Snel, F., Gruys, E., Niv, D., Batt, R.M., Munn, E.A., Buttress, N. and Pepys, M.B., 1984. Isolation and characterization of C-reactive protein from the dog. Immunology, 53, 307– 313

    PubMed  CAS  Google Scholar 

  • Caspi, D., Snel, F.W.J.J., Batt, R.M., Bennett, D., Rutteman, G.R., Hartman, E.G., Baltz Marilyn L., Gruys, E. and Pepys, M.B., 1987. C-reactive protein in dogs. American Journal of Veterinary Research, 48, 919–921

    PubMed  CAS  Google Scholar 

  • Conner, J.G., Eckersall, P.D., Ferguson, J. and Douglas, T.A., 1988. Acute phase response in the dog following surgical trauma. Research in Veterinary Science, 45, 107–110

    PubMed  CAS  Google Scholar 

  • De Winter, R.J., Fischer, J., Bholasingh, R., van Straalen, J.P. and de Jong, T., 2000. C-reactive protein and cardiac troponin T in risk stratification: differences in optimal timing of tests early after the onset of chest pain. Clinical Chemistry, 46, 1597–1603

    PubMed  CAS  Google Scholar 

  • Eckersall, P.D., Conner, J.G. and Parton, H., 1989. An enzyme-linked immunosorbent assay for canine C-reactive protein. Veterinary Record, 124, 490–491

    PubMed  CAS  Google Scholar 

  • Eckersall, P.D., Conner, J.G. and Harvie, J., 1991. An immunoturbidimetric assay for canine C-reactive protein. Veterinary Research Communications, 15, 17–24

    Article  PubMed  CAS  Google Scholar 

  • Eckersall, P.D., Duthie, S., Toussaint, M.J.M., Gruys, E., Heegaard, P., Alava, M., Lipperheide, C. and Madec, F., 1999a. Standardization of diagnosis assays for animal acute phase protein. Advances in Veterinary Medicine, 41, 643–655

    Article  CAS  Google Scholar 

  • Eckersall, P.D., Duthie, S., Safi, S., Moffatt, D., Horadagoda, N.U., Doyle, S., Parton, R., Bennett, D. and Fitzpatrick J.L., 1999b. An automated biochemical assay for haptoglobin: prevention of interference from albumin. Comparative Haematology International, 9, 117–124

    Article  CAS  Google Scholar 

  • Erkens, J.H.F., Dieleman, S.J., Dressendörfer, R.A. and Strasburger, C.J., 1998. A time-resolved fluoroimmunoassay for cortisol in unextracted bovine plasma or serum with optimized procedures to eliminate steroid binding protein interference and to minimize non-specific streptavidin–europium binding. The Journal of Steroid Biochemistry and Molecular Biology, 67, 153–161

    Article  PubMed  CAS  Google Scholar 

  • Fiet, J., Boudi, A., Giton, F., Villette, J.M., Boudu, Ph., Soliman, H., Morineau, G. and Galons, H., 2000. Plasma 21-deoxycortisol: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunoassay using 125iodine. The Journal of Steroid Biochemistry and Molecular Biology, 72, 55–60

    Article  PubMed  CAS  Google Scholar 

  • Hayashi, S., Jinbo, T., Iguchi, K., Shimizu, M., Shimada, T., Nomura, M., Ishida, Y. and Yamamoto, S., 2001. A comparison of the concentrations of C-reactive protein and α1-acid glycoprotein in the serum of young and adult dogs with acute inflammation. Veterinary Research Communications, 25, 117–120

    Article  PubMed  CAS  Google Scholar 

  • Jergens, A.E., Schreiner, C.A., Frank, D.E., Niyo, Y., Ahrens, F.E., Eckersall, P.D., Benson, T.J. and Evans, R., 2003. A scoring index for disease activity in canine inflammatory bowel disease. Journal of Veterinary Internal Medicine, 17, 291–297

    Article  PubMed  Google Scholar 

  • Kimura, H., Mukaida, M., Wang, G., Yuan, J. and Matsumoto, K., 2000. Dual-label time-resolved fluoroimmunoassay of psychopharmaceuticals and stimulants in serum. Forensic Science International, 113, 345–351

    Article  PubMed  CAS  Google Scholar 

  • Kjelgaard-Hansen, M., Kristensen, A.T. and Jensen, A.L., 2003a. Evaluation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of C-reactive protein in canine serum. Journal of Veterinary Medicine A, 50, 164–168

    CAS  Google Scholar 

  • Kjelgaard-Hansen, M., Jensen, A.L. and Kristensen, A.T., 2003b. Evaluation of a commercially available human C-reactive protein (CRP) turbidometric immunoassay for determination of canine serum CRP concentration. Veterinary Clinical Pathology, 32, 81–87

    Google Scholar 

  • Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685

    Article  PubMed  CAS  Google Scholar 

  • Lövgren, T. and Pettersson, P., 2000. Time-resolved fluoroimmunoassay, advantages and limitations. In: K. Van Dyke and R. Van Dyke (eds). Luminescence Immunoassay and Molecular Applications, (CRC Press, Boca Raton, FL), 233–253

    Google Scholar 

  • Marténez-Subiela, S., Tecles, F., Eckersall, P.D. and Cerón, J.J., 2002. Serum concentrations of acute phase proteins in dogs with leishmaniasis. Veterinary Record, 150, 241–244

    Google Scholar 

  • McGrotty, Y., McComb, C., Ramsey, I.K. and Eckersall, P.D., 2001. Evaluation of a rapid assay for canine C-reactive protein. Veterinary Clinical Pathology, 30, 164

    Google Scholar 

  • Ndung'u, J.M., Eckersall, P.D. and Jennings, F.W., 1991. Elevation of the concentration of acute phase proteins in dogs infected with Trypanosoma brucei. Acta Tropica, 49, 77–86

    Article  PubMed  Google Scholar 

  • Onishi, T., Shimizu, T. and Kajikawa, T., 1994. Simple and efficient purification of C-reactive protein from canine serum. The Journal of Veterinary Medical Science, 56, 417–419

    PubMed  CAS  Google Scholar 

  • Otabe, K., Sugimoto, T., Jinbo, T., Honda, M., Kitao, S., Hayashi, S., Shimizu, M. and Yamamoto, S., 1998. Physiological levels of C-reactive protein in normal canine sera. Veterinary Research Communications, 22, 77–85

    Article  PubMed  CAS  Google Scholar 

  • Otabe, K., Ito, T., Sugimoto, T. and Yamamoto, S., 2000. C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury. Laboratory Animals, 34, 434–438

    PubMed  CAS  Google Scholar 

  • Parra, M.D., Tuomola, M., Cabezas-Herrera, J. and Cerón, J.J., 2005. Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood. American Journal of Veterinary Research, 66, 62–66

    Article  PubMed  CAS  Google Scholar 

  • Pontet, M., Engler, R. and Jayle, M.F., 1978. One step preparation of both human C-reactive protein and Cit. FEBS Letters, 88, 172–175

    Article  PubMed  CAS  Google Scholar 

  • Rikihisa, Y., Yamamoto, S., Kwak, I., Iqbal, Z., Kociba, G., Mott, J. and Chichanarisiwithaya, W., 1994. C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis. Journal of Clinical Microbiology, 32, 912–917

    PubMed  CAS  Google Scholar 

  • Tagata, K., Yokoyama, S., Ginbo, T., Honda, M., Okimura, T., Odakura, M., Nomura, M. and Yamamoto, S., 1996. Quantitative capillary reversed passive latex agglutination test for C-reactive protein (CRP) in the dog. Veterinary Research Communications, 20, 21–30

    Article  PubMed  CAS  Google Scholar 

  • Tarkkinen, P., Palenius, T. and Lövgren, T., 2002. Ultrarapid, ultrasensitive one-step kinetic immunoassay for C-reactive protein (CRP) in whole blood samples: measurement of the entire CRP concentration range with a single sample dilution. Clinical Chemistry, 48, 269–277

    PubMed  CAS  Google Scholar 

  • Wergeland, R., Oyen, O., Bentland, O. and Stokke, O., 1999. MicroCRP: a highly sensitive CRP method applied in the monitoring of renal allograft recipients. Scandinavian Journal of Clinical and Laboratory Investigation, 59, 405–416

    PubMed  CAS  Google Scholar 

  • Yamamoto, S., Abe, N., Santsuka, H., Shida, T., Kishida, K, Kuwajima, S., Yamada, M., Morimatsu, M. and Naiki, M., 1993a. Efficient preparation of monospecific anti-canine C-reactive protein serum and purification of canine C-reactive protein by affinity chromatography. Veterinary Immunology and Immunopathology, 36, 293–301

    CAS  Google Scholar 

  • Yamamoto, S., Shida, T., Miyaji. S., Santsuka, H., Fujise, H., Mukawa, K., Furukawa, E., Nagae, T. and Nauki, M., 1993b. Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas. Veterinary Research Communications, 17, 85–93

    CAS  Google Scholar 

  • Yamamoto, S., Shida, T., Okimura, T., Otabe, K., Honda, M., Ashida, Y., Furukawa, E., Sarikaputi, M. and Naiki, M., 1994. Determination of C-reactive protein in serum and plasma from healthy dogs and dogs with pneumonia by ELISA and slide reversed passive latex agglutination test. The Veterinary Quarterly, 16, 74–77

    PubMed  CAS  Google Scholar 

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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

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  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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