P3: Day 2 Keynote Address By: Jack Levin, MD The Limulus Amebocyte Lysate (LAL) Test: From Past to Present Jack Levin, M.D. Depts. Of Laboratory Medicine and Medicine Univ. of California School of Medicine San Francisco, CA P3: Day 2 Keynote Address The American Horseshoe Crab (Limulus polyphemus) By: Jack Levin, MD P3: Day 2 Keynote Address By: Jack Levin, MD P3: Day 2 Keynote Address Jack Levin at the Marine Biological Laboratory 1973 By: Jack Levin, MD P3: Day 2 Keynote Address Blood Obtained by Cardiac Puncture and Dripped Directly into Collection Fluid Copper‐Containing Hemocyanin is the Oxygen Carrying Pigment in Limulus Blood By: Jack Levin, MD P3: Day 2 Keynote Address Amebocytes Settling Washed and Resuspended Limulus Amebocytes By: Jack Levin, MD P3: Day 2 Keynote Address By: Jack Levin, MD Limulus Amebocyte Lysate (Gel Clot Endpoint) Gel Floc. Control + Endotoxin Reaction between Endotoxin and Coagulation System is Enzymatic P3: Day 2 Keynote Address By: Jack Levin, MD Increase in Light Scattering After Addition of Increasing Concentrations of Endotoxin to Amebocyte Lysate Increase in Optical Density After Addition of Different Concentrations of Endotoxin P3: Day 2 Keynote Address By: Jack Levin, MD Increase in Light Scattering Following Addition of Bacterial Endotoxin to LAL µg/ml Levin. J Lab Clin Med 75:903, 1970 Basic Endpoints for the Limulus Amebocyte Lysate Test for Bacterial Endotoxins Sensitivity(EU/ml) Gel Clot Turbidimetric/Kinetic Chromogenic/Kinetic 0.03 0.001‐0.005 0.005 P3: Day 2 Keynote Address First Demonstration of an Enzymatic Blood Coagulation Mechanism in an Invertebrate Blood Coagulation in Limulus is Enzymatic By: Jack Levin, MD P3: Day 2 Keynote Address By: Jack Levin, MD P3: Day 2 Keynote Address By: Jack Levin, MD Gelation of Limulus Amebocyte Lysate by E. coli or Klebsiella Endotoxin vs Rabbit Pyrogenicity E n d o t o x i n Gelation Time increased as the Concentration of Endotoxin Decreased E. coli Kleb. Arrows indicate Gelation Times of Endotoxin Concentrations that produced Pyrogenic Response in 50% of Rabbits Applications of the Limulus Amebocyte Lysate Test Pharmaceutical Preparations In Process Monitoring Large volume parenterals Water Supplies Small volume parenterals Medical Devices Drugs (parenteral) Body Fluids Vaccines Blood Biological products Spinal Fluid (CSF) Radioisotopes Urine Plasma Protein Fractions Environment Food Quality Air Quality Space P3: Day 2 Keynote Address Warning that the Biological Activities of many Reagents may actually be due to contamination by Bacterial Endotoxin (1984) By: Jack Levin, MD P3: Day 2 Keynote Address By: Jack Levin, MD Effect of Contamination by Endotoxin (LPS) on the Biological Activity of Erythropoietin (EP) LPS‐Free LPS‐Free Zuckerman. Blood 54:146, 1979 Erythropoietin Preparations had been Contaminated with Bacterial Endotoxins P3: Day 2 Keynote Address Correlation between Plasma LPS Levels and Mortality in Systemic Meningococcal Disease Correlation between Detection of LPS with Limulus Test and Gas Chromatography‐ Mass Spectrometry By: Jack Levin, MD P3: Day 2 Keynote Address Measurement of N. meningitidis in Plasma (DNA vs LAL) ØvstebØ. J Clin Microbiol 42:2980, 2004 Berman. J Ped 88:553, 1976 By: Jack Levin, MD P3: Day 2 Keynote Address Measurement of N. meningitidis in CSF (DNA vs LAL) ØvstebØ. J Clin Microbiol 42:2980, 2004 Amebocyte Lysate Test 1. Adequately sensitive 2. Correlates with other bioassays for endotoxin 3. Correlates with number of copies of DNA 4. Multiple endpoints By: Jack Levin, MD P3: Day 2 Keynote Address Inhibition of Detection of Endotoxin by Human Serum Levin. J Lab Clin Med 75:903,1970 Chloroform Extraction of LPS from Plasma Tomasulo. J Lab Clin Med 75:903, 1970 By: Jack Levin, MD P3: Day 2 Keynote Address By: Jack Levin, MD Comparison of Plasma Treatment Methods to Remove Inhibition of LAL Dilution/Heating TFA Acid Oxidation CHCl3 Roth. J Lab Clin Med 116: 153, 1990 Effect of Hemoglobin on Electrophoretic Mobility of LPS (PAGE) LPS Hb + LPS LPS Kaca. J Biol Chem 269:25078, 1994 P3: Day 2 Keynote Address By: Jack Levin, MD Effect of Hemoglobin on Density of Lipopolysaccharide Hb + LPS LPS Alone LPS Kaca. J Biol Chem 209:25078, 1994 Hemoglobin Produces Disaggregation of LPS P3: Day 2 Keynote Address By: Jack Levin, MD Disaggregation of Lipopolysaccharide by Hemoglobin A. Aggregated S. minnesota 595 B. Disaggregated after Incubation with Hgb (18 Hr/37C) C. Heterogeneous E. coli O55:B5 D. Disaggregated after incubation with Hgb (18 Hr/37C) X 180,000 + Hgb + Hgb Roth. in Brade. Endotoxin in Health and Disease 1999 Shortening of Gelation Time (LAL) by Increasing Concentrations of Hemoglobin Roth. Transf 33:919, 1993 P3: Day 2 Keynote Address Hemoglobin Increases Mortality Associated with Endotoxemia S u r v i v a l ααHb + LPS Su. Infect Immun 65:1258, 1997 Bacterial Lipopolysaccharide (LPS) Chemical Structure By: Jack Levin, MD P3: Day 2 Keynote Address Lipid A Chemical Structure Endotoxin is a Component of the Bacterial Cell Wall By: Jack Levin, MD P3: Day 2 Keynote Address Inhibition of the LAL Assay (Selected Examples) Chelators Citrate, EDTA Low pH (LAL optimum 7.2) High Ionic Strength Proteins (Non‐Specific Binding) Cationic (+ve) Proteins Assay Tube Composition/Surface Characteristics Failure to Sonicate and/or Vortex Bolden. Bacterial Endotoxin Summit 2014 By: Jack Levin, MD P3: Day 2 Keynote Address Platco. Bacterial Endotoxin Summit 2014 Comparison of LAL and Rabbit Pyrogen Tests for Endotoxin (Total Performed) Rabbit Pyrogen Tests 28,410 LAL (BET) Tests 143,196 Positive Tests detected by either procedure 37 Positive BET Test 37 Positive USP Rabbit Test 4 USP Rabbit Positive/BET Neg. 0 Mascoli. in Biomed Appl Horseshoe Crab. p. 387, 1979 By: Jack Levin, MD P3: Day 2 Keynote Address Red Knots weigh between 90 and 120 grams on arrival at Delaware Bay, but after a few weeks of feeding on crab eggs, they nearly double in weight By: Jack Levin, MD P3: Day 2 Keynote Address The American Horseshoe Crab CN Shuster Jr, RB Barlow and HJ Brockmann, Eds. Harvard Univ. Press, Cambridge, 2003 By: Jack Levin, MD
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