Endotoxin detection in a competitive electrochemical assay: synthesis of a suitable endotoxin conjugate.

A biotin-lipopolysaccharide (biotin-LPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng(-1). The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotin-LPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidin-horseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotin-LPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml(-1), equivalent to 0.07 EU ml(-1).

[1]  N. Santos,et al.  Evaluation of Lipopolysaccharide Aggregation by Light Scattering Spectroscopy , 2003, Chembiochem : a European journal of chemical biology.

[2]  K. V. van Holde,et al.  Physical studies of lysozyme. I. Characterization. , 1962, The Journal of biological chemistry.

[3]  J. W. Whittaker,et al.  Kinetic isotope effects as probes of the mechanism of galactose oxidase. , 1998, Biochemistry.

[4]  D. Wild.  The Immunoassay Handbook , 2001 .

[5]  J. Shands,et al.  The dispersion of gram-negative lipopolysaccharide by deoxycholate. Subunit molecular weight. , 1980, The Journal of biological chemistry.

[6]  K. Brandenburg,et al.  Physicochemical properties of bacterial glycopolymers in relation to bioactivity. , 2003, Carbohydrate research.

[7]  L R WETTER,et al.  Immunological studies on egg white proteins. IV. Immunochemical and physical studies of lysozyme. , 1951, The Journal of biological chemistry.

[8]  R. Liddington,et al.  Crystal structure of an endotoxin‐neutralizing protein from the horseshoe crab, Limulus anti‐LPS factor, at 1.5 A resolution. , 1993, The EMBO journal.

[9]  R. Canfield.  THE AMINO ACID SEQUENCE OF EGG WHITE LYSOZYME. , 1963, The Journal of biological chemistry.

[10]  J. V. Alstine,et al.  Protein-rejecting ability of surface-bound dextran in end-on and side-on configurations: comparison to PEG. , 1995, Journal of biomedical materials research.

[11]  B. Lindner,et al.  Molecular structure of lipid A, the endotoxic center of bacterial lipopolysaccharides. , 1994, Advances in carbohydrate chemistry and biochemistry.

[12]  Johan Kuiper,et al.  Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock , 2003, Clinical Microbiology Reviews.

[13]  Chaby.  Strategies for the control of LPS-mediated pathophysiological disorders. , 1999, Drug discovery today.

[14]  T. Kirikae,et al.  The chemical structure of bacterial endotoxin in relation to bioactivity. , 1993, Immunobiology.

[15]  C A Aurell,et al.  Critical aggregation concentrations of gram-negative bacterial lipopolysaccharides (LPS). , 1998, Biochemical and biophysical research communications.

[16]  J. Luk,et al.  Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells. , 1995, Analytical biochemistry.

[17]  T. Ogawa,et al.  Requirement of a properly acylated beta(1-6)-D-glucosamine disaccharide bisphosphate structure for efficient manifestation of full endotoxic and associated bioactivities of lipid A , 1987, Infection and immunity.

[18]  M. Wilchek,et al.  Foreword and introduction to the book (strept)avidin-biotin system. , 1999, Biomolecular engineering.

[19]  N. Surolia,et al.  Kinetics and Mechanism of the Recognition of Endotoxin by Polymyxin B , 1998 .

[20]  C. Danilowicz,et al.  Osmium complexes bearing functional groups : building blocks for integrated chemical systems , 1998 .

[21]  K. Triantafilou,et al.  Lipopolysaccharide (LPS) labeled with Alexa 488 hydrazide as a novel probe for LPS binding studies. , 2000, Cytometry.

[22]  N. Green,et al.  AVIDIN. 1. THE USE OF (14-C)BIOTIN FOR KINETIC STUDIES AND FOR ASSAY. , 1963, The Biochemical journal.

[23]  H. S. Warren,et al.  Binding and neutralization of endotoxin by Limulus antilipopolysaccharide factor , 1992, Infection and immunity.

[24]  B. Monks,et al.  Lysines 128 and 132 Enable Lipopolysaccharide Binding to MD-2, Leading to Toll-like Receptor-4 Aggregation and Signal Transduction* , 2003, Journal of Biological Chemistry.

[25]  T. McIntosh,et al.  Lipopolysaccharide bilayer structure: effect of chemotype, core mutations, divalent cations, and temperature. , 1999, Biochemistry.

[26]  M. Bolognesi,et al.  The X-ray three-dimensional structure of avidin. , 1999, Biomolecular engineering.

[27]  J. Schneider-Mergener,et al.  High Affinity Endotoxin-binding and Neutralizing Peptides Based on the Crystal Structure of Recombinant Limulus Anti-lipopolysaccharide Factor* , 1996, The Journal of Biological Chemistry.

[28]  R Polzius,et al.  Optimization of biosensing using grating couplers: immobilization on tantalum oxide waveguides. , 1996, Biosensors & bioelectronics.

[29]  H. Odeberg,et al.  Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. , 1978, The Journal of biological chemistry.

[30]  J. Hasday,et al.  Endotoxin-Neutralizing Protein Protects against Endotoxin-Induced Endothelial Barrier Dysfunction , 1998, Infection and Immunity.

[31]  F B Anspach,et al.  Endotoxin removal from protein solutions. , 2000, Journal of biotechnology.

[32]  J. Ghrayeb,et al.  Lipopolysaccharide (LPS) Neutralizing Peptides Reveal a Lipid A Binding Site of LPS Binding Protein (*) , 1995, The Journal of Biological Chemistry.

[33]  J. Savéant,et al.  Quantitative analysis of catalysis and inhibition at horseradish peroxidase monolayers immobilized on an electrode surface. , 2003, Journal of the American Chemical Society.

[34]  U. Jönsson,et al.  Real-time biospecific interaction analysis , 1992 .

[35]  F. Battaglini,et al.  Use of an antimicrobial protein for endotoxin detection in a competitive electrochemical assay. , 2005, Analytical chemistry.