HEK-Blue™ LPS Detection Kit 2

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Cell-based colorimetric assay for the detection of biologically active endotoxin
HEK-Blue™ LPS Detection Kit 2

The HEK-Blue™ LPS Detection Kit is based on the ability of TLR4 to recognize structurally different LPS from gram-negative bacteria and in particular lipid A, their toxic moiety. Proprietary cells engineered to become extremely sensitive to LPS, called HEK-Blue™-4 cells, are the main feature of this endotoxin detection kit. The presence of very low concentrations of LPS, starting as low as 0.03 ng/ml, are detected by the HEK-Blue™-4 cells leading to the activation of NF-κB. Using HEK-Blue™ Detection, a specific detection medium, NF-κB activation can be observed with the naked eye or quantified by reading the OD at 650 nm.

The HEK-Blue™ LPS Detection Kit 2 is an assay intended for the detection and quantification of biologically active LPS for research purposes.

It is based on the activation of Toll-like receptor (TLR) 4, the mammalian endotoxin sensor (Beutler B., 2002). TLR4 recognizes structurally different LPS from gram-negative bacteria.

Proprietary cells engineered to become extremely sensitive to LPS, called HEK-Blue™-4 cells, are the main feature of this endotoxin detection kit. These cells stably express human TLR4 and an NF-kB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.

The presence of minute quantities of LPS, starting as low as 0.01 EU/ml, are detected by the HEK-Blue™-4 cells leading to the activation of NF-kB.

Using QUANTI-Blue™, a SEAP detection medium that produces a purple/blue color, NF-kB activation can be observed with the naked eye or measured at 620-655 nm. Since the absorbance is in direct proportion to the amount of endotoxin present, the concentration of endotoxin can be calculated from a standard curve obtained using serial dilutions of the HEK-Blue™ Endotoxin Standard (a preparation of E. coli 055:B5 LPS standardized against FDA approved control standard endotoxin (CSE)).

References:

  1. Zhang G. and S. Ghosh, 2000. Molecular mechanisms of NF-kappaB activation induced by bacterial lipopolysaccharide through Toll-like receptors. J Endotoxin Res. 6(6): p. 453-7.
  2. Beutler B., 2003. Science review: key inflammatory and stress pathways in critical illness - the central role of the Toll-like receptors. Crit Care. 7(1): p. 39-46.
  3. Dobrovolskaia M.A. and S.N. Vogel, 2002. Toll receptors, CD14, and macrophage activation and deactivation by LPS. Microbes Infect. 4(9): p. 903-14.
  4. Wolk K. et al., 2000.Impaired antigen presentation by human monocytes during endotoxin tolerance. Blood. 96(1): p. 218-23.
  5. Kariko K. et al., 2004. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J Immunol. 172(11):6545-9.
  6. Darveau RP et al., 2004. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4. Infect Immun. 72(9):5041-51.

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