Integrated strategy for the separation of endotoxins from biofluids. LPS capture on newly synthesized protein

Endotoxin, or lipopolysaccharide (LPS), is recognized as the most powerful microbial mediator implicated in the pathogenesis of sepsis, a life-threatening organ disfunction whose overall prognosis is still poor. Therefore, the need to detect and remove LPS has led some authors to focus their efforts on the design of pathogen cleansing microdevices. Micromagnetophoretic cleansing involves a first step for the selective binding and sequestration of LPS on non-toxic particles followed by the removal of the loaded particles from the fluid medium. While outstanding progress has been made in magnetic separation, the state of the art of the first step still needs to fill important gaps in knowledge to advance towards the development of detection systems. Thus, this work reports an integrated methodology to advance the design of the LPS sequestration stage to promote its separation from biofluids. The methodology combines protein and separation fundamentals and starts with the synthesis of an anti LPS factor protein (LALF), followed by the quantitative determination of its binding strength to LPS. For this analysis LALF was supported on agarose beads and the variables affecting LPS binding, as binding and capture temperature, the optimum bead:protein and protein:LPS ratios, have been experimentally studied. The methodology and results here reported constitute the information needed to advance the knowledge for the correct design of LPS separation devices.