Case report: How researchers used our TNF-α ELISA to evaluate endotoxin contaminations

Before Bengt Fadeel’s group could examine the biological effects of their materials of interest (graphene-based materials), they were challenged by how to evaluate their toxicology as they had reasons to suspect they could interfere with assays typically used for cytotoxicity or endotoxin testing. The study by Mukherjee et al. (2016) describes how they judged their setup aimed to differentiate between intrinsic immunomodulatory effects and contaminations from bacterial endotoxins. The authors named the assay TNF-α expression test (TET).  

In brief, they stimulated primary human monocyte-derived macrophages (HMDM) in the presence or absence of the specific LPS inhibitor, polymyxin B sulfate, and analyzed supernatants with Mabtech’s Human TNF-α ELISA. LPS was included as a positive control and different LPS titrations were used for the standard curve. As a prerequisite for this test, non-toxic doses of the test material were established in a cell viability assay. The authors conclude that their TNF-α assay shows a sensitivity comparable to the Limulus amebocyte lysate (LAL) assay they ran in parallel, which, however, showed interference due to internal properties of the test material.  

Using an ELISA makes this setup a quantitative method. In addition, the authors argue that using HMDMs makes the method more sensitive than monocyte activation tests (MAT) based on monocytic cell lines. Furthermore, by including the specific LPS inhibitor, the authors obtained a functional assay to differentiate between endotoxin contaminations and intrinsic pro-inflammatory properties. 

Please consult the original publication for the authors’ recommendation on how they set up their TNF-α test. 

 

Having validated a tool detecting endotoxin contaminations on their research materials, the research group could proceed and investigate any immunomodulatory effect of graphene oxide. In a recent publication (Peng et al. 2023), the group demonstrated the effect of graphene oxide on the microbiome eliciting a type 2 immune response in zebrafish.