Comparison of methods for depletion of albumin and IgG from equine serum.
Abstract: Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One-dimensional gel electrophoresis followed by densitometry was used to determine percent of albumin, IgG, and both in pooled serum from 3 horses before and after application of 7 depletion methods. Repeatability was determined by applying the 2 best methods to serum samples from 6 grade horses. Results: For pooled serum, depletion rates varied from 35-90% for albumin and 0-94% for IgG. In the repeatability study, the ProteoExtract method combined with protein G Sepharose beads to remove additional IgG provided the best overall performance with 66% albumin depletion and 100% IgG depletion. A protocol using protein G Sepharose beads to remove IgG followed by ethanol precipitation of nonalbumin proteins with albumin remaining in the supernatant was the second most effective, with 85% albumin depletion and 55% IgG depletion. Although a multiprotein immunodepletion column effectively removed 90% of the albumin, the method was ineffective at removing IgG. Conclusions: Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.
©2010 American Society for Veterinary Clinical Pathology.
Publication Date: 2010-08-18 PubMed ID: 20727125DOI: 10.1111/j.1939-165X.2010.00241.xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research article focuses on various methods for reducing the presence of albumin and IgG proteins in horse serum, in order to allow for easier detection of disease biomarkers. The results revealed considerable differences in efficacy and a combination of the ProteoExtract kit and manual incubation with protein G Sepharose beads was found to be the most effective.
Objective and Methods
- The main aim of this study was to assess the ability of multiple commercially available kits and procedures to deplete albumin and IgG, two highly present proteins in equine serum, that might overshadow the presence of low concentration disease biomarkers.
- Depletion rates were assessed using one-dimensional gel electrophoresis and densitometry on pooled serum from three horses. The seven proposed depletion methods were compared.
- The repeatability of the two most effective methods was then tested on serum samples from six different horses.
Results of Depletion Methods
- The rates at which albumin and IgG were removed varied across the different methods. Albumin was reduced between 35-90% and IgG between 0-94% in pooled serum.
- The most effective method was the ProteoExtract kit combined with Protein G Sepharose beads for additional IgG removal. This method achieved 66% albumin and 100% IgG depletion.
- The second most effective method was Protein G Sepharose beads for IgG removal, followed by nonalbumin protein precipitation with ethanol, with the remaining albumin stayed in the supernatant, achieving 85% albumin depletion and 55% IgG depletion.
- Although multiprotein immunodepletion column use removed 90% of the albumin, this method was less effective at removing IgG.
Conclusions
- Kits designed to remove albumin and IgG from human serum have varying efficiency levels when applied to equine serum.
- The best overall performance was achieved by combining the ProteoExtract kit with manual incubation using Protein G Sepharose beads, suggesting this could be a preferred methodology for such procedures in the future.
Cite This Article
APA
Olver CS, Webb TL, Long LJ, Scherman H, Prenni JE.
(2010).
Comparison of methods for depletion of albumin and IgG from equine serum.
Vet Clin Pathol, 39(3), 337-345.
https://doi.org/10.1111/j.1939-165X.2010.00241.x Publication
Researcher Affiliations
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1691, USA. colver@colostate.edu
MeSH Terms
- Animals
- Biomarkers / blood
- Blood Proteins / analysis
- Blood Proteins / isolation & purification
- Densitometry / methods
- Densitometry / veterinary
- Electrophoresis, Agar Gel / methods
- Electrophoresis, Agar Gel / veterinary
- Female
- Horses / blood
- Immunoglobulin G / blood
- Immunoglobulin G / isolation & purification
- Male
- Reproducibility of Results
- Sensitivity and Specificity
- Serum Albumin / analysis
- Serum Albumin / isolation & purification
Citations
This article has been cited 4 times.- Rowland AL, Burns ME, Levine GJ, Watts AE. Preparation Technique Affects Recipient Immune Targeting of Autologous Mesenchymal Stem Cells. Front Vet Sci 2021;8:724041.
- Rowland AL, Miller D, Berglund A, Schnabel LV, Levine GJ, Antczak DF, Watts AE. Cross-matching of allogeneic mesenchymal stromal cells eliminates recipient immune targeting. Stem Cells Transl Med 2021 May;10(5):694-710.
- Poltep K, Tesena P, Yingchutrakul Y, Taylor J, Wongtawan T. Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses. J Equine Sci 2018 Sep;29(3):53-60.
- Malik A, Al-Senaidy A, Skrzypczak-Jankun E, Jankun J. Isolation and characterization of serum albumin from Camelus dromedarius. Exp Ther Med 2013 Aug;6(2):519-524.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
