OJVM  Vol.3 No.6 , October 2013
Initial Validation of Cytokine Measurement by ELISA in Canine Feces
Abstract: Measurement of fecal cytokines has been used as a marker of intestinal inflammation in people and correlates with endoscopic findings. The aim of this study was to evaluate the use of canine-specific enzyme-linked immunosorbant assays (ELISAs) for quantification of cytokines in canine fecal samples as a non-invasive biomarker. Interleukin (IL)-6, -8, -10, -23/12p40 and TNF-a were assessed by using spiked fecal samples from 3 healthy dogs. Standard curve validation was performed, and the impact of time to freeze, duration of storage and number of freeze-thaw cycles on cytokine concentration were also examined. All the cytokines assayed could be detected, with varying accuracy. The mean coefficient of variation (CV) for all standard curves ranged from 2.95%-9.8%. The mean intra-assay CV ranged from 3.1%-11.14%, and inter-assay CV from 4.36%-18.83%. Recovery of IL-23 was poor (7.23%-17.12%), precluding further interpretation of stability studies. Mean recovery did not appear to be affected by time to freeze and repeat freeze-thaw cycles in all cytokines investigated. Recovery for all cytokines after short-term storage of 30 days at -80℃ showed a recovery of <70% or >130%. In conclusion, although fecal IL-6, -8, -10, and TNF-a could be used as biomarkers of intestinal inflammation in the dog, the quality of laboratory performance and poor recovery at lower concentrations limit their application. Bench-top and freeze-thaw stability was acceptable, and samples should ideally be analyzed within a week. Investigation involving dogs with acute and chronic inflammatory intestinal disease is required to determine the role of this methodology in a clinical setting.  
Cite this paper: N. Prakash, P. Stumbles and C. Mansfield, "Initial Validation of Cytokine Measurement by ELISA in Canine Feces," Open Journal of Veterinary Medicine, Vol. 3 No. 6, 2013, pp. 282-288. doi: 10.4236/ojvm.2013.36046.

[1]   M. D. Willard, A. E. Jergens, R. B. Duncan, M. S. Leib, M. D McCracken, R. C. DeNovo, et al., “Interobserver Variation among Histopathologic Evaluations of Intestinal Tissues from Dogs and Cats,” Journal of the American Veterinary Medical Association, Vol. 220, No. 8, 2002, pp. 1177-1182.

[2]   M. D. Willard, S. L. Lovering, N. D. Cohen and B. R. Weeks, “Quality of Tissue Specimens Obtained Endoscopically from the Duodenum of Dogs and Cats,” Journal of the American Veterinary Medical Association, Vol. 219, No. 4, 2001, pp. 474-479.

[3]   M. D. Willard, G. E. Moore, B. D. Denton, M. J. Day, J. Mansell, T. Bilzer, et al., “Effect of Tissue Processing on Assessment of Endoscopic Intestinal Biopsies in Dogs and Cats,” Journal of Veterinary Internal Medicine, Vol. 24, No. 1, 2010, pp. 84-89.

[4]   S. B. Hanauer, “Inflammatory Bowel Disease,” The New England Journal of Medicine, Vol. 334, No. 13, 1996, pp. 841-848.

[5]   K. Allenspach, B. Wieland, A. Grone and F. Gaschen, “Chronic Enteropathies in Dogs: Evaluation of Risk Factors for Negative Outcome,” Journal of Veterinary Internal Medicine, Vol. 21, No. 4, 2007, pp. 700-708.

[6]   A. E. Jergens, C. A. Schreiner, D. E. Frank, Y. Niyo, F. E. Ahrens, P. D. Eckersall, et al., “A Scoring Index for Disease Activity in Canine Inflammatory Bowel Disease,” Journal of Veterinary Internal Medicine, Vol. 17, No. 3, 2003, pp. 291-297.

[7]   A. Poullis, R. Foster, T. C. Northfield and M. A. Mendall, “Review Article: Faecal Markers in the Assessment of Activity in Inflammatory Bowel Disease,” Alimentary Pharmacology & Therapeutics, Vol. 16, No. 4, 2002, pp. 675-681.

[8]   I. Angriman, M. Scarpa, R. D’Incà, D. Basso, C. Ruffolo, L. Polese, et al., “Enzymes in Feces: Useful Markers of Chronic Inflammatory Bowel Disease,” Clinica Chimica Acta, Vol. 381, No. 1, 2007 pp. 63-68.

[9]   K. F. Murphy, A. J. German, C. G. Ruaux, J. M. Steiner, D. A. Williams and E. J. Hall, “Fecal Alpha1-Proteinase Inhibitor Concentration in Dogs with Chronic Gastrointestinal Disease,” Veterinary Clinical Pathology, Vol. 32, No. 2, 2003, pp. 67-72.

[10]   C. G. Ruaux, J. M. Steiner and D. A. Williams, “Protein-Losing Enteropathy in Dogs is Associated with Decreased Fecal Proteolytic Activity,” Veterinary Clinical Pathology, Vol. 33, No. 1, 2004, pp. 20-22.

[11]   N. Berghoff and J. M. Steiner, “Laboratory Tests for the Diagnosis and Management of Chronic Canine and Feline Enteropathies,” Veterinary Clinics of North America: Small Animal, Vol. 41, No. 2, 2011, pp. 311-328.

[12]   T. Melgarejo, D. A. Williams and E. K. Asem, “Enzyme-Linked Immunosorbent Assay for Canine A1-Protease Inhibitor,” American Journal of Veterinary Research, Vol. 59, No. 2, 1998, pp. 127-130.

[13]   H. Lettesjo, T. Hansson, C. Peterson, K. A. Ung, G. Ringstrom, H. Abrahamsson, et al., “Detection of Inflammatory Markers in Stools from Patients with Irritable Bowel Syndrome and Collagenous Colitis,” Scandinavian Journal of Gastroenterology, Vol. 41, No. 1, 2006, pp. 54-59.

[14]   C. P. Braegger, S. Nicholls, S. H. Murch, S. Stephens and T. T. MacDonald, “Tumour Necrosis Factor Alpha in Stool as a Marker of Intestinal Inflammation,” Lancet, Vol. 339, No. 8785, 1992, pp. 89-91.

[15]   C. G. Peterson, P. Sangfelt, M. Wagner, T. Hansson, H. Lettesjo and M. Carlson, “Fecal Levels of Leukocyte Markers Reflect Disease Activity in Patients with Ulcerative Colitis,” Scandinavian Journal of Clinical & Laboratory Investigation, Vol. 67, No. 8, 2007, pp. 810-820.

[16]   T. Saiki, K. Mitsuyama, A. Toyonaga, H. Ishida and K. Tanikawa, “Detection of Pro-and AntiInflammatory Cytokines in Stools of Patients with Inflammatory Bowel Disease,” Scandinavian Journal of Gastroenterology, Vol. 33, No, 6, 1998, pp. 616-622.

[17]   G. Ko, Z. D. Jiang, P. C. Okhuysen and H. L. DuPont, “Fecal Cytokines and Markers of Intestinal Inflammation in International Travelers with Diarrhea Due to Noroviruses,” Journal of Medical Virology, Vol. 78, No. 6, 2006, pp. 825-828.

[18]   D. E. Greenberg, Z. D. Jiang, R. Steffen, M. P. Verenker and H. L. DuPont, “Markers of Inflammation in Bacterial Diarrhea among Travelers with a Focus on Enteroaggregative Escherichia Coli Pathogenicity,” The Journal of Infectious Diseases, Vol. 185, No. 7, 2002, pp. 944-949.

[19]   S. Ardizzone and G. B. Porro, “Inflammatory Bowel Disease: New Insights into Pathogenesis and Treatment,” Journal of Internal Medicine, Vol. 252, No. 6, 2002, pp. 475-496.

[20]   I. Monteleone, F. Pallone and G. Monteleone, “Interleu-kin-23 and Th17 Cells in the Control of Gut Inflammation,” Mediators of Inflammation, Vol. 2009, 2009, Article ID: 297645.

[21]   P. J. Morrison, S. J. Ballantyne and M. C. Kullberg, “Interleukin-23 and T Helper 17-Type Responses in Intestinal Inflammation: From Cytokines to T-Cell Plasticity,” Immunology, Vol. 133, No. 4, 2011, pp. 397-408.

[22]   US Department of Health and Human Services Food and Drug Administration, “Guidance for Industry: Bioanalytical Method Validation,” Rockville, 2001.

[23]   M. A. Valentin, S. Ma, A. Zhao A, F. Legay and A. Avrameas, “Validation of Immunoassay for Protein Biomarkers: Bioanalytical Study Plan Implementation to Support Preclinical and Clinical Studies,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 55, No. 5, 2011, pp. 869-877.

[24]   M. A. Sidler, S. T. Leach and A. S. Day, “Fecal S100a12 and Fecal Calprotectin as Noninvasive Markers for Infammatory Bowel Disease in Children,” Inflammatory Bowel Diseases, Vol. 14, No. 3, 2008, pp. 359-366.

[25]   J. W. Lee, V. Devaranarayan, Y. C. Barrett, R. Weiner, J. Allinson, S. Fountain, et al., “Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement,” Pharmaceutical Research, Vol. 23, No. 2, 2006, pp. 312-328.

[26]   K. Brookes, J. Cummings, A. Backen, A. Greystoke, T. Ward, G. C. Jayson, et al., “Issues on Fit-For-Purpose Validation of a Panel of ELISAs for Application as Biomarkers in Clinical Trials of Anti-Angiogenic Drugs,” British Journal of Cancer, Vol. 102, No. 10, 2010, pp. 1524-1532.

[27]   A. E. Jergens, I. M. Sonea, A. M. O’Connor, L. K. Kauffman, S. D. Grozdanic, M. R. Ackermann, et al., “Intestinal Cytokine mRNA Expression in Canine Inflammatory Bowel Disease: A Meta-Analysis with Critical Appraisal,” Comp Med, Vol. 59, No. 2, 2009, pp. 153-162.

[28]   N. Nguyen Van, K. Taglinger, C. R. Helps, S. Tasker, T. J. Gruffydd-Jones and M. J. Day, “Measurement of Cytokine mRNA Expression in Intestinal Biopsies of Cats with Inflammatory Enteropathy Using Quantitative Real-Time RT-PCR,” Veterinary Immunology and Immunopathology, Vol. 113, No. 3-4, 2006, pp. 404-414.

[29]   I. R. Peters, C. R. Helps, E. L. Calvert, E. J. Hall and M. J. Day, “Cytokine mRNA Quantification in Histologically Normal Canine Duodenal Mucosa by Real-Time RT-PCR,” Veterinary Immunology and Immunopathology, Vol. 103, No. 1-2, 2005, pp. 101-111.

[30]   A. J. German, C. R. Helps, E. J. Hall and M. J. Day, “Cytokine mRNA Expression in Mucosal Biopsies from German Shepherd Dogs with Small Intestinal Enteropathies,” Digestive Diseases and Sciences, Vol. 45, No. 1, 2000, pp. 7-17.

[31]   A. E. Ridyard, T. J. Nuttall, R. W. Else, J. W. Simpson and H. R. Miller, “Evaluation of Th1, Th2 and Immunosuppressive Cytokine mRNA Expression within the Colonic Mucosa of Dogs with Idiopathic Lymphocytic-Plas-macytic Colitis,” Veterinary Immunology and Immunopathology, Vol. 86, No. 3-4, 2002, pp. 205-214.

[32]   I. R. Peters, C. R. Helps, E. L. Calvert, E. J. Hall and M. J. Day, “Cytokine mRNA Quantification in Duodenal Mucosa from Dogs with Chronic Enteropathies by Real-Time RT-PCR,” Journal of Veterinary Internal Medicine, Vol. 19, No. 5, 2005, pp. 644-653.

[33]   M. Sarra, F. Pallone, T. T. MacDonald and G. Montele-one, “Il-23/Il-17 Axis in IBD,” Inflammatory Bowel Diseases, Vol. 16, No. 10, 2010, pp. 1808-1813.

[34]   W. Shen and S. K. Durum, “Synergy of Il-23 and Th17 Cytokines: New Light on Inflammatory Bowel Disease,” Neurochemical Research, Vol. 35, No. 6, 2010, pp. 940-946.

[35]   H. H. Uhlig, B. S. McKenzie, S. Hue, C. Thompson, B. Joyce-Shaikh, R. Stepankova, et al., “Differential Activity of Il-12 and Il-23 in Mucosal and Systemic Innate Immune Pathology,” Immunity, Vol. 25, No. 2, 2006, pp. 309-318.

[36]   W. De Jager, K. Bourcier, G. T. Rijkers, B. J. Prakken and V. Seyfert-Margolis, “Prerequisites for Cytokine Measurements in Clinical Trials with Multiplex Immunoassays” BMC Immunology, Vol. 10, 2009, pp. 52-63.

[37]   N. Aziz, P. Nishanian, R. Mitsuyasu, R. Detels and J. L. Fahey, “Variables That Affect Assays for Plasma Cytokines and Soluble Activation Markers” Clinical and Diagnostic Laboratory Immunology, Vol. 6, No. 1, 1999, pp. 89-95.

[38]   B. DeSilva, W. Smith, R. Weiner, M. Kelley, J. Smolec, B. Lee, et al., “Recommendations for the Bioanylytical Method Validation of Ligand Binding Assays to Support Pharmacokinetic Assessments of Macromolecules,” Pharmaceutical Research, Vol. 20, No. 11, 2003, pp. 1885-1900.