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Endotoxin contamination in ovalbumin as viewed from a nano‐immunotherapy perspective

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Abstract Ovalbumin (OVA) is a model antigen commonly incorporated in smartly designed nanoparticles for delivery into antigen‐presenting cells (APC), aiming to investigate the immune activity and therapeutic efficacy of nanoparticles that contain immunoregulatory compounds. However, the immunoresponse observed in nano‐immunotherapy may unexpectedly arise from endotoxin impurity of OVA in the nanoparticles. Literature review shows that most researchers did not notice the importance of endotoxin‐free OVA when used in nano‐immunotherapy studies. Concentration at as low as 5 μg/ml OVA from Sigma‐Aldrich (contains 0.625 ng/ml endotoxin) was able to activate APC such as dendritic cells and macrophages. Here, we proposed that the endotoxin impurity in OVA or the finished nanoproducts should be determined by both Limulus Amebocyte Lysate (LAL) and cell‐based assay, to ensure the endotoxin‐free quality of the nanoparticles. The endotoxin in OVA can be removed by endotoxin removal column and phase separation methods and endotoxin‐free OVA can be purchased. This perspective alerts the researchers of endotoxin impurity of OVA that may transfer into the finished nanoparticles and introduce an unfavorable immunoregulatory function with false‐positive results. OVA with minimal endotoxin level should be used in nano‐immunotherapy studies to accurately reflect the true effects of nanoparticles on the immune system. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Immunostimulatory effects induced by endotoxin contamination of OVA protein detected by flow cytometry. (a) Protein expression of costimulatory molecule CD86 of DC and macrophage was significantly enhanced through TLR4 pathway by OVA from Sigma but not Hyglos. DC (differentiated from bone marrow mesenchymal stem cells by GM‐CSF/IL‐4) and macrophage were treated with OVA (50 μg/ml) for 24 h. Sigma‐OVA treated DC/macrophage was also incubated with TLR4 antagonist (resatorvid, 1 μg/ml). (b) SIINFEKL epitope in the context of H‐2 kb on the surface of DC was quantified using PE‐tagged monoclonal antibody 25‐D1.16. MFI indicated mean fluorescence intensity. (c) SIINFEKL epitope induced by Sigma‐OVA at different concentrations from 5 to 100 μg/ml. Statistical significance was assessed using one‐way ANOVA. Data represent mean ± SD. **p < 0.01, ***p < 0.001
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Literature summary for immunotherapeutic related work associated with OVA containing nanoparticles. (a) Summary of 155 literature sources involving OVA nanoparticles. A literature search using keywords of “ovalbumin” and “nanoparticles” was performed and a totally 155 research articles were obtained from seven major journals featured with nanomedicine (Biomaterials, Advanced Functional Materials‐AFM, ACS Nano, ACS Applied Materials & Interfaces‐ACS AMI, Journal of Controlled Release‐JCR, International Journal of Nanomedicine, Advanced Materials‐AM) (108 papers found) and Google Scholar (47 papers obtained from other journals). These research articles were selected because their work used OVA as a raw material in preparing nanoparticles for immunotherapeutic studies. (b) Summary of OVA suppliers summarized from the above‐mentioned literature
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Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

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