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Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats

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  • 1 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 2 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 3 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 4 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 5 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 6 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 7 Department of Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

Abstract

OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples.

SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats.

PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups.

RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection.

CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations.

Abstract

OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples.

SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats.

PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups.

RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection.

CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations.

Contributor Notes

Dr. Halpin's present address is 3964 Red Bank Rd, Cincinnati, OH 45227.

Dr. Saunders’ present address is 625 Harrison St, West Lafayette, IN 47907.

Dr. Thompson's present address is 7385 S Buffalo Dr, Las Vegas, NV 89113.

Dr. Rohde Newgent's present address is 1 Pine St, Neptune City, NJ 07753.

Address correspondence to Dr. DeClue (DeClueA@missouri.edu).