Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches
Abstract
:1. Introduction
2. Results
2.1. Purity of IgG Samples
2.2. Impurities Identification
2.3. IgG Thermal Stabiliy
2.4. Aggregate Content
2.5. IgG subclass Distribution
2.6. Vaa Venom-Specific Protective Efficacy
3. Discussion
3.1. Protein A Affinity Chromatography
3.2. Precipitation-Based Purification Procedures
3.3. Ion-Exchange Chromatography
3.4. Thermal Stability
3.5. IgG Subclass Redistribution in Relation to Venom-Neutralising Activity
4. Conclusions
5. Materials and Methods
5.1. Animals, Snake Venom, Plasma Pool, and Reagents
5.2. IgG Purification Procedures
5.2.1. Caprylic Acid Precipitation
5.2.2. Ammonium Sulphate Precipitation
5.2.3. Anion-Exchange Chromatography
5.2.4. Cation-Exchange Chromatography
5.2.5. Protein A Affinity Chromatography
5.3. Diafiltration
5.4. Thermal Shift Assay
5.5. Purity and Aggregate Content Profiling
5.6. Mass Spectrometry for Protein Identification
5.7. Protein and IgG Content Determination
5.8. IgG Subclass Quantity Determination
5.9. Lethal Toxicity Neutralization Assay
5.10. Data Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Purification Method | IgG Purity (%) |
---|---|
ASP1 | 83.1 ± 1.5 (n = 2) |
ASP2 | 96.6 ± 1.2 (n = 4) |
AEX1 | 76.7 ± 0.8 (n = 2) |
AEX2 | 98.0 ± 0.8 (n = 4) |
CEX1 | 72.6 ± 2.2 (n = 3) |
CEX2 | 97.8 ± 0.8 (n = 4) |
CAP | 93.3 ± 1.4 (n = 4) |
AC | 92.1 ± 1.7 (n = 4) |
Purification Method | Aggregate Content (%) |
---|---|
ASP1 | 4.5 ± 1.4 |
ASP2 | 1.4 ± 0.2 |
AEX1 | 0.0 ± 0.0 |
AEX2 | 0.4 ± 0.1 |
CEX1 | 5.4 ± 0.9 |
CEX2 | 1.2 ± 0.3 |
CAP | 2.3 ± 0.3 |
AC | 6.4 ± 1.4 |
Hyperimmune Horse Plasma | CEX2 | AC | CAP | |
---|---|---|---|---|
R/LD50 mL−1 | 23.89 | 14.15 | <5.48 | 12.05 |
γ(IgG)/mg mL−1 | 22.89 | 31.40 | 13.35 | 12.84 |
Specific activity [R/γ(IgG)] | 1.03 | 0.45 | <0.41 | 1.07 |
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Mateljak Lukačević, S.; Kurtović, T.; Lang Balija, M.; Brgles, M.; Steinberger, S.; Marchetti-Deschmann, M.; Halassy, B. Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches. Toxins 2020, 12, 798. https://doi.org/10.3390/toxins12120798
Mateljak Lukačević S, Kurtović T, Lang Balija M, Brgles M, Steinberger S, Marchetti-Deschmann M, Halassy B. Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches. Toxins. 2020; 12(12):798. https://doi.org/10.3390/toxins12120798
Chicago/Turabian StyleMateljak Lukačević, Sanja, Tihana Kurtović, Maja Lang Balija, Marija Brgles, Stephanie Steinberger, Martina Marchetti-Deschmann, and Beata Halassy. 2020. "Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches" Toxins 12, no. 12: 798. https://doi.org/10.3390/toxins12120798
APA StyleMateljak Lukačević, S., Kurtović, T., Lang Balija, M., Brgles, M., Steinberger, S., Marchetti-Deschmann, M., & Halassy, B. (2020). Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches. Toxins, 12(12), 798. https://doi.org/10.3390/toxins12120798