A Biomarker for Copper Associated Hepatitis in Labrador Retrievers

Introduction
Copper toxicosis is a hereditary disease in the Labrador retriever. Due to a disturbed copper metabolism, copper accumulates in the liver, leading to liver damage (1). Cur- rently, the only way to establish the diagnosis is by means of a liver biopsy. therefore, a non-invasive biomarker is needed. two promising biomarkers in erythrocytes are CCS and SoD1, which are known to reflect copper status in mice and rat (2,3). However, CCS and SoD1 antibodies are not validated for use in canine erythrocytes. therefore the aim of this project is to validate these antibodies for future use as a biomarker for copper toxicosis in the dog.

Material and Methods
Western blots for the detection of CCS and SoD1 in dog erythrocytes was optimized using different antibody dilutions and protein concentrations. the specificity of anti- bodies for CCS and SoD1 was confirmed with sirna knock-down in canine bile-duct epithelial cells. Knockdown of CCS and SoD1 was confirmed using qpCr and Western blot.

Results
Western blots on erythrocyte proteins indicated a single band at 35 kDa for CCS and 23 kDa for SoD1. optimal primary antibody dilution for Western blot is 1:1,000 and optimal protein concentration is 20μg for SoD1 and 60μg for CCS. Knock-down for CCS and SoD1 mrna levels was 93 and 73 percent, respectively. Western blot confirmed the knock-down given by the disappearance of the immunoreactive band for CCS and SoD1.

Conclusion
Antibodies for CCS and SoD1 are applicable for use in canine erythrocytes.

Practical relevance
Erythrocyte CCS and SoD1 levels will be tested as a potential biomarker of copper toxi- cosis in the Labrador retriever. if they show a correlation with hepatic copper, they will be used to identify dogs at risk, so illness can be prevented. Moreover, this biomarker could be useful in monitoring copper status during and after treatment.