Microfiltration method of removal of bacterial contaminants and their monitoring by nitric oxide and Limulus assays

Similar to lipopolysaccharide (LPS), a product of Gram-negative bacteria, the signal macromolecules of Gram-positive bacteria lipoteichoic acid (LTA) and peptidoglycan (PGN) possess multiple biological activities. They may be a source of misinterpretation of experimental findings. We have found that not only LPS but also LTA and PGN can be detected by the Limulus amebocyte lysate (LAL) assay. All of them stimulate the high output in vitro nitric oxide (NO) production of in rat peritoneal cells. The onset of the NO enhancement was observed with 25-100 pg/ml of LPS and 25-100 ng/ml of PGN and LTA. Polymyxin B (PMX), if applied at concentration 10,000-fold higher than that of LPS, can completely inhibit the NO and LAL binding responses of LPS. The NO-stimulatory and LAL-binding properties of LTA and PGN are not eliminated by PMX. Handling of LPS contamination with PMX may be associated with serious problems because it possesses intrinsic biological activity and becomes cytotoxic at concentration >25 mu g/ml. The present findings suggest a convenient alternative avoiding these issues. As monitored by the NO and LAL assays, even high amounts of LPS as well as PGN and LTA can be removed by molecular mass cutoff microfiltration. All types of the filters (3 kDa to 100 kDa) are equally effective. It is suggested that the microfiltration procedure may be considered as a preferable, general and easy method of sample decontamination. (C) 2012 Elsevier Inc. All rights reserved.