Enzyme-Linked Immunosorbent Assay to Quantitate Serum Ferritin in Black and White Ruffed Lemurs (Varecia Variegata Variegata)

Abstract

Lemurs in captivity progressively accumulate iron deposits in a variety of organs (hemosiderosis) including duodenum, liver, and spleen throughout their lives. When excessive, the toxic effects of intracellular iron on parenchymal cells, particularly the liver, can result in clinical disease and death. The pathogenesis of excessive iron storage in these species has been attributed to dietary factors related to diets commonly fed in captivity. Tissue iron stores can be directly estimated by tissue biopsy and histologic examination, or quantitated by chemical analysis of biopsy tissue, However, expense and risk associated with anesthesia and surgery prevent routine use of tissue biopsy to assess iron status. A noninvasive means of assessing total body iron stores is needed to monitor iron stores in lemurs to determine whether dietary modification is preventing excessive iron deposition, and to monitor potential therapies such as phlebotomy or chelation. Serum ferritin concentration correlates with tissue iron stores in humans, horses, calves, dogs, cats, and pigs. Serum ferritin is considered the best serum analyte to predict total body iron stores in these species and is more reliable than serum iron or total iron binding capacity, both of which may be affected by disorders unrelated to iron adequacy or excess including hypoproteinemia, chronic infection, hemolytic anemia, hypothyroidism, renal disease, and drug administration. We have developed an enzyme-linked immunosorbent assay to measure serum ferritin in lemurs. The assay uses polyclonal rabbit anti-human ferritin antibodies in a sandwich arrangement. Ferritin isolated from liver and spleen of a black and white ruffed lemur (Varecia variegata variegata) was used as a standard. Ferritin standards were linear from 0 to 50 microg/L. Recovery of purified ferritin from lemur serum varied from 95% to 110%. The within-assay variability was 4.5%, and the assay-to-assay variability for three different samples ranged from 10% to 17%. The assay also measures serum ferritin in several other lemur species.

Evaluation of iron status in lemurs by analysis of serum iron and ferritin concentrations, total iron-binding capacity, and transferrin saturation

“…Additionally, the capture and indicator antibodies used in the assay are polyclonal and should, therefore, recognize multiple epitopes. 20 For these reasons, the developers of the assay are confident that it reliably measures ferritin in a variety of lemur taxa. Nonetheless, it is prudent to interpret the ferritin measurements obtained in taxa other than ruffed lemurs with some caution until the assay can be independently validated in each species.…”

“…g Ferritin measurements were performed by use of a quantitative ELISA developed at that laboratory for detection of lemur ferritin. 20 Percent transferrin saturation was calculated as SI concentration/TIBc X 100. All samples were analyzed in a single-batch assay to minimize variability.…”

Survey and Clinical Application of Serum Iron, Total Iron Binding Capacity, Transferrin Saturation, and Serum Ferritin in Captive Black and White Ruffed Lemurs (Varecia Variegata Variegata)

“…Serum ferritin concentration was measured with a recently developed enzyme-linked immunosorbent assay using polyclonal anti-human ferritin antibodies adapted to measure black and white ruffed lemur ferritin (Kansas State University). 1 For the analysis of data, no assumption of normal distribution was made and nonparametric statistical analysis was utilized. 17 Correlations between factors were assessed using the Spearman correlation coefficient ().…”