Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay

Abstract

Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production

“…It is recommended to be applied for clarification in case of a discrepancy between results of different LAL assay formats as it is regarded as being the most robust and least prone to interference, as optical interference can be ruled out, since the detection of endotoxins is based on visual inspection of gel clot formation. Nevertheless, we previously noted that the LAL gel clot assay showed significant interference with iron oxide-silica-core shell nanoparticles as well as other types of nanoparticles, leading either to assay enhancement or inhibition [26]. The GO dispersions were found to be endotoxin positive in the LAL gel clot assay at concentrations as low as 7.8 μg/ml (GO-A), 31.25 μg/ml (GO-B), 15.625 μg/ml (GO-C), and 1.95 μg/ml (GO-D), respectively (Table 3).…”

“…Importantly, the European Directive 2010/63/EU on the protection of animals used for scientific purposes enforces the replacement of animal tests when validated alternatives exist. While the LAL assay is known to be very sensitive, several laboratories have reported problems of interference of nanoparticles with one or more of the LAL assay formats [12, 15–18]. Previous studies have suggested that TLR4 reporter cells could be used to evaluate endotoxin contamination of metal/metal oxide nanoparticles [17].…”

Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

“…It has been shown that the gel clot LAL assay is not accurate for testing endotoxin contamination in particles, while the chromogenic LAL assay showed higher sensitivity and no interference (46). The unsuitability of the gel clot assay has also been shown for silica, silver (Ag), titanium dioxide, calcium carbonate, and other clinical-grade NPs (37, 38, 47), suggesting that the gel clot assay should not be used for testing endotoxin in ENM in general. However, despite these new evidences, the use of the gel clot assay is still recommended in a FDA guidance document to solve discrepancies between results from different LAL formats in industry (48).…”

Graphene and the Immune System: A Romance of Many Dimensions

“…Importantly, the European Directive 2010/63/EU on the protection of animals used for scientific purposes enforces the replacement of animal tests when validated alternatives exist. While the LAL assay is known to be very sensitive, several laboratories have reported problems of interference of various types of nanomaterials with one or more of the LAL assay formats (20–22). Indeed, carbon-based nanomaterials including GBMs were shown to interfere with the LAL assay, which may lead to erroneous results or mask the effects of the materials themselves (23, 24).…”