Lipopolysaccharide interferes with the use of the human Cell Line Activation Test to determine the allergic potential of proteins

Abstract

It was believed that high molecular weight molecules including proteins cannot penetrate the skin. However, protein penetration through disrupted/ruptured skin has been reported recently, thus carrying the potential for inducing an allergic response. We used the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, to assess the allergic potential of proteins by measuring levels of CD86 and CD54 in the human monocytic leukemia cell line THP-1. Six allergens including ovalbumin (OVA) and human serum albumin (HSA; negative control) upregulated CD86 and/or CD54; a false-positive result was obtained using HSA. This was caused by lipopolysaccharide (LPS) contamination. Naturally derived materials often include LPS at various concentrations and may influence protein induction of CD86 and CD54. Additionally, polymyxin B, an LPS inhibitor, could not completely overcome the effect of LPS. Therefore, if test proteins contain ≥0.1 EU/mL LPS, their allergenic potency will not be assessed accurately using h-CLAT. These data show that naturally occurring materials or those derived from living organisms should be evaluated for their LPS content. It is important to confirm the applicability of in vitro methods such as h-CLAT for assessing the allergenic potency of naturally occurring proteins; our findings can be a foundation for future studies.