Enzyme-linked immunosorbent assay to measure serum ferritin in toucans (Ramphastidae sp.)

Master of ScienceDepartment of Diagnostic Medicine/ PathobiologyLisa M. PohlmanBackground: Iron storage disease has proven to be a serious health concern for captive toucans. Physiologic mechanisms to efficiently extract iron from naturally iron-deficient diets appear the likely cause of iron overload when fed iron-sufficient diets in captivity. Iron overload can result in diabetes, heart failure, and even death. Serum ferritin concentrations are considered the most reliable screening tool to predict total body iron stores in many species, but an assay has not been available to measure serum ferritin in toucans. Objective: The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum ferritin in toucans using a polyclonal antibody in a sandwich arrangement. Methods: Ferritin was isolated from toucan liver and used as a standard. A rabbit polyclonal anti-toucan antibody was used as the capture antibody and as a detection antibody conjugated to horseradish peroxidase. Linearity of toucan ferritin standards, effect of serum dilution, recovery of added ferritin standards, and intra- and inter-assay variability were determined. Results: Ferritin standards were linear from 0 to 50 ng/ml. The relationship between serum dilution and serum ferritin concentration was also linear. When 10, 20, 30, 40, or 50 ng/ml of purified toucan ferritin were added to diluted serum, the recoveries varied from 69% to 104%. The intra-assay variability for four test serum samples averaged 11% and the inter-assay variability for the same four samples averaged 11%. Conclusions: Although the results from the linearity and recovery studies are promising for assay development when viewed independently, preliminary ferritin concentrations from all toucans studied are much higher than expected. Upon further evaluation including Dot blot assays, Western blot assays, SDS-PAGE, and protein determination of the ferritin stock solution, it was determined that the ferritin stock solution did not contain a pure protein and therefore likely renders the assay invalid. Further testing is needed to confirm these findings

ASVCP guidelines : principles of quality assurance and standards for veterinary clinical pathology (version 3.0) : developed by the American Society for Veterinary Clinical Pathology's (ASVCP) Quality Assurance and Laboratory Standards (QALS) Committee

This guideline is a revision of the previous (finalized 2009) document of the same name, American Society for Veterinary Clinical Pathology's Principles of Quality Assurance and Standards for Veterinary Clinical Pathology, developed by the Quality Assurance and Laboratory Standards (QALS) committee (and colloquially known as the “general guideline”), archived on the ASVCP website (www.asvcp.org/page/QALS_Guidelines), on a newly established Wiley freeware page (https://onlinelibrary.wiley.com/page/journal/1939165x/homepage/Qals) as published in Veterinary Clinical Pathology in three sectional special reports. This guideline's intended audiences are professional veterinary laboratorians (pathologists, technologists/technicians, research scientists, and pathology residents), operators/users of in‐clinic instruments/in‐clinic laboratories, and more broadly, all producers and consumers of clinical veterinary laboratory data, namely the veterinarians/training veterinarians who have the responsibility of ordering appropriate tests and properly interpreting results that inform further diagnostic and treatment decisions.Supplement material 1: Section 10S Supplemental Information on Immunoassay TechniquesSupplement material 2: Checklist for ASVCP Quality AssuranceGuideline Section 2, Total QualityManagement System (TQMS)(v.3, 2019)Supplement material 3: Checklist for Checklist for ASVCP Quality Assurance Guideline Section 3, Preanalytical Factors Important in Veterinary Clinical Pathology (v.3, 2019)Supplement material 4: Checklist for ASVCP Quality Assurance Guideline Section 4, Analytical factors Important in Veterinary Clinical Pathology (v.3, 2019)Supplement material 5: Checklist for ASVCP Quality Assurance Guideline Section 5, Hematology (v.3, 2019)Supplement material 6: Checklist for ASVCP Quality Assurance Guideline Section 6, HemostasisTesting (v.3, 2019)Supplement material 7: Checklist for Guideline Section 7, CrossmatchingSupplement material 8: Checklist for ASVCP Quality Assurance Guideline Section 8, Urinalysis (v.3, 2019)Supplement material 9: Checklist for ASVCP Quality Assurance Guideline Section 9, Cytology, Fluid Analysis, and Immunocytochemistry (v.3, 2019)Supplement material 10: Checklist for ASVCP Quality Assurance Guideline Section 10, Endocrinology and Immunoassays (v.3, 2019)Supplement material 11: Checklist for ASVCP Quality Assurance Guideline Section 11, Protein electrophoresis and Electrophoresis-based Immunotyping (v.3, 2019)Supplement material 12: Checklist for ASVCP Quality Assurance Guideline Section 12, Postanalytical Factors Important in Veterinary Clinical Pathology(v.3, 2019)http://wileyonlinelibrary.com/journal/vcphj2020Companion Animal Clinical Studie