Biomedical Applications of Limulus Amebocyte Lysate

  • Thomas J. NovitskyEmail author


This year celebrates the 30th anniversary of the licensing of Limulus amebocyte lysate (LAL) by the US Food and Drug Administration (FDA) as a test for the presence of endotoxin in biologicals, pharmaceutical drugs, and medical devices. LAL is currently recognized by several major pharmacopoeias and is used worldwide. That a suitable alternative for the detection of endotoxin has not supplanted LAL is indicative of its superior reliability. Since its discovery, LAL has proven its usefulness not only to detect harmful levels of endotoxin (as pyrogens) in pharmaceutical products, but has become an indispensable tool in controlling endotoxin in processes and equipment used to produce pharmaceuticals. Indeed, the exquisite sensitivity of LAL compared to other assays for endotoxin/pyrogen has proven extremely useful in monitoring high-purity water used as a prime ingredient or processing agent for all biologicals, drugs, and devices. LAL has also become the assay of choice for researchers studying both the clinical and the environmental effects of endotoxin. To highlight its usefulness, various specific applications of LAL including modifications of the assay to allow testing of complex substances will be described. Finally, although horseshoe crab mortality associated with LAL production is low, the LAL industry has taken steps to find a synthetic substitute and to produce reagents and methods that use much less LAL than traditional assays. That the horseshoe crab has uniquely contributed a test that profoundly affects the safety of pharmaceuticals should be celebrated and rewarded by continuing to protect this valuable resource.


Cotton Fiber Horseshoe Crab Limulus Amebocyte Lysate United States Pharmacopoeia Limulus Amebocyte Lysate Assay 
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  1. Alpert G, Baldwin G, Thompson C, Wainwright NR, Novitsky TJ, Gillis Z, Siber GR, Parsonnet J, Fleisher GR (1992) Limulus antilipopolysaccharide factor protects rabbits from meningococcal endotoxin shock. J Infect Dis 165:494–500PubMedCrossRefGoogle Scholar
  2. Andrä J, Garidel P, Majerle A, Jerala R, Ridge R, Paus E, Novitsky T, Koch MHJ, Brandendurg K (2004) Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide. Eur J Biochem 271: 2037–2046PubMedCrossRefGoogle Scholar
  3. Arduino MJ. Bland LA, Tipple MA, Aguero SM, Favero MS, Jarvis WR (1989) Growth and endotoxin production of Yersinia enterocolitica and Enterobacter agglomerans in packed erythrocytes. J Clin Microbiol 27:1483–1485PubMedGoogle Scholar
  4. Armstrong PB (2003) Chapter 12. Internal defense against pathogenic invasion: The immune system. In: Shuster CN Jr., Barlow RB, Brockman HJ (eds) The American Horseshoe Crab. Harvard University Press, Cambridge, MA, pp 288–309Google Scholar
  5. Associates of Cape Cod, Inc. (2007) Fungitell™ Assay.
  6. Barthel KW (1990) 4 Palaeoenvironment and sedimentation. In: Solnhofen: A study in Mesozoic paleontology. Morris SC (ed) Swinburne NHM (transl) Cambridge University Press, Cambridge, UK, pp 56–67Google Scholar
  7. Bates DW, Parsonnet, J, Ketchum, PA, Miller, EB, Novitsky, TJ, Sands, K, Hibberd, PL, Graman, PS, Lanken, PN, Schwartz, JS, Kahn, K, Snydman, DR, Moore, R, Black, E, Platt, R, AMCC Sepsis Project Working Grp (1998) Limulus amebocyte lysate assay for detection of endotoxin in patients with sepsis syndrome. Clin Infect Dis 27:582–591PubMedCrossRefGoogle Scholar
  8. Case MJ, Ryther SS, Novitsky TJ (1983) Detection of endotoxin in antibiotic solutions with Limulus amoebocyte lysate. Antimicrob Agents Chemother 23:649–652PubMedCrossRefGoogle Scholar
  9. Charles River Laboratories (2007) Endosafe®-PTS™ Completes Journey to the International Space Station.
  10. Cooper JE, Levin J, Wagner NH Jr. (1970) New rapid in vitro test for pyrogen in short-lived radiopharmaceuticals. J Nucl Med 11–301Google Scholar
  11. Dawson ME, Novitsky TJ, Gould MJ (1988) Microbes, endotoxins and water. Pharm Engineer 8(2):9–12Google Scholar
  12. Ding JL, Chai A, Pui WM, Jo B (1999) Expression of full length and deletion homologues of Carcinoscorpius rotundicauda Factor C in Saccharomyces cerevisiae: immunoreactivity and endotoxin binding. J Endotoxin Res 4:33–43Google Scholar
  13. European Pharmacopoeia Commission (1987) Bacterial endotoxins. In: European Pharmacopoeia Vol 2.1.9Google Scholar
  14. Evans TM, Schllinger JE, Stuart TG (1978) Rapid determination of bacteriological water quality by using Limulus lysate. Appl Environ Microbiol 35:376–382PubMedGoogle Scholar
  15. Garcia C, Saladino R, Thompson C, Hammer B, Parsonnet J, Wainwright N, Novitsky T, Fleisher GR, Siber G (1994) Effect of a recombinant endotoxin-neutralizing protein on endotoxin shock in rabbits. Crit Care Med 22:1211–1218PubMedCrossRefGoogle Scholar
  16. Hasday JD, Bascom R, Costa JJ, Fitzgerald T, Dubin W (1999) Bacterial endotoxin is an active component of cigarette smoke. Chest 115:829–835PubMedCrossRefGoogle Scholar
  17. Hobbie JE, Novitsky TJ, Rublee PA, Fergusen RL, Palumbo AV (1987) Microbiology. In: Backus RH (ed), Georges Bank. MIT Press, Cambridge, pp 247–251Google Scholar
  18. Hochstein HD, Novitsky TJ (2003) Limulus Endotoxins Test, In: Easter M (ed) Rapid Microbiological Methods. Interpharm Press, IL pp 187–210CrossRefGoogle Scholar
  19. Jay JM (1997) The Limulus lysate endotoxin assay as a test of microbial quality of ground beef. J Appl Bacteriol 43:99–109Google Scholar
  20. Jorgensen JH (1986) Clinical applications of the Limulus amebocyte lysate test. Chapter 4. In: Handbook of endotoxin Vol 4 Clinical aspects of endotoxin shock. Procter RA (ed) Elsevier, Amsterdam pp 127–153Google Scholar
  21. Jorgensen JH, Lee JC, Alexander GA, Wolf HW (1979) Comparison of Limulus assay, standard plate count, and total coliform count for microbiological assessment of renovated wastewater. Appl Environ Microbiol 37:928–931PubMedGoogle Scholar
  22. Ketchum PA, Parsonnet J, Stotts LS, Novitsky TJ, Schlain B, Bates DW, AMCC SEPSIS Project (1997) Utilization of a chromogenic Limulus amebocyte lysate blood assay in a multi-center study of sepsis. J Endotoxin Res 4:9–16Google Scholar
  23. Kuppermann N, Nelson DS, Saladino RA, Thompson CM, Sattler F, Novitsky TJ, Fleisher GR, Siber GR (1994) Comparison of a recombinant endotoxin-neutralizing protein with a human monoclonal antibody to endotoxin for the treatment of Escherichia coli sepsis in rats. J Infect Dis 170:630–635PubMedCrossRefGoogle Scholar
  24. Levin J, Hochstein HD, Novitsky TJ (2003) Chapter 13. Clotting cells and Limulus amebocyte lysate: An amazing analytical tool. In: Shuster CN Jr., Barlow RB, Brockman HJ (eds) The American Horseshoe Crab. Harvard University Press, Cambridge, MA, pp 310–334Google Scholar
  25. Lonza (2007) Endotoxin & Microbial Detection.
  26. Mikolajcik EM, Brucker RB (1983) Limulus amebocyte lysate assay – A rapid test for thee assessment of raw and pasteurized milk quality. Dairy Food Sanit 3:129–131Google Scholar
  27. Milton DK, Gould MC, Roslansky PF, Novitsky TJ (1987) New estimates of endotoxin content of cotton dust: SDS suspension and kinetic LAL test. In: Rylander R, Burrell R, Peterson Y (eds) Endotoxin Inhalation Workshop, Proc of Beltwide Cotton Prod ConfGoogle Scholar
  28. Miyazaki Y, Kohno S, Koga H, Kaku M, Mitsutake K, Maesaki S, Yasuoka A, Hana S, Tanaka S, Tamura H (1992) G Test, a new direct method for diagnosis of Candida infection: comparison with assays for beta-glucan and mannan antigen in a rabbit model of systemic candidiasis. J Clin Lab Anal 6:315–318PubMedCrossRefGoogle Scholar
  29. Morita T, Tanaka S, Nakamura T, Iwanaga S (1981) A new (1–3)-ß-d-glucan-mediated coagulation pathway found in Limulus amebocytes. FEBS Lett 129:318–321CrossRefGoogle Scholar
  30. Mottar J (1987) A colorimetric endotoxin assay for the determination of the bacteriological quality of milk. Neth Milk Dairy J 41:137–145Google Scholar
  31. Nelson D, Kuppermann N, Fleisher GR, Hammer BK, Thompson CM, Garcia CT, Novitsky TJ, Parsonnet J, Onderdonk A, Siber GR (1995) Recombinant endotoxin neutralizing protein improves survival from Escherichia coli sepsis in rats. Crit Care Med 23:92–98PubMedCrossRefGoogle Scholar
  32. Novitsky TJ (1982a) Limulus amebocyte lysate: life saving extract from the horseshoe crab. Aquasphere, J New Engl Aquarium 16:26–31Google Scholar
  33. Novitsky TJ (1982b) Living fossil: it helps to reduce pyrogen risk. Dial Transplan Today Nov:1011, 1020Google Scholar
  34. Novitsky TJ (1984) Monitoring and validation of high purity water systems with the Limulus amebocyte lysate test for pyrogens. Pharm Eng 4:21–33Google Scholar
  35. Novitsky TJ (1987) Bacterial endotoxins (pyrogens) in purified waters. In: Mittelman MM, Geesey GG (eds) Biological fouling of industrial water systems: A problem solving approach. Water Micro Associates, San Diego, pp 77–96Google Scholar
  36. Novitsky TJ (1988) Industrial application of the LAL test. Rapid detection of bacteria and bacterial endotoxins. In: Metodi rapidi ed automatizzati nella microbiologia applicata. Societa editoriale farmaceutica, Milano. pp179–180Google Scholar
  37. Novitsky TJ (1994) Limulus amebocyte lysate (LAL) detection of endotoxin in human blood. J Endotoxin Res 1:253–263Google Scholar
  38. Novitsky TJ (1996) Endotoxin: Does its detection provide diagnostic benefit? In: Faist E, Baue AE, Schildberg FW (eds) The immune consequences of trauma, shock and sepsis, Mechanisms and therapeutic approaches. Vol. II. Pabst Science Publishers, Zagreb. pp 171–182Google Scholar
  39. Novitsky TJ (1999) Endotoxin detection in body fluids: Chemical versus bioassay methodology. In: Brade H, Opal SM, Vogel SN, Morrison DC (eds) Endotoxin in Health and Disease, Marcel Dekker, Inc New York-Basel, pp 831–839Google Scholar
  40. Novitsky TJ (2001) Biomedical products from the horseshoe crab: Lessons for sound resource management and conservation. In: Tanacredi JT (ed) Limulus in the Limelight. Kluwer/Plenum Publishing, New York, pp 79–84Google Scholar
  41. Novitsky TJ, Roslansky PF, Siber GR, Warren HS (1985) Turbidimetric method for quantifying serum inhibition of Limulus amoebocyte lysate. J Clin Microbiol 20:211–216Google Scholar
  42. Novitsky TJ, Siber GR, Ketchum P (1996) Limulus anti-LPS factors. In: Faist E, Baue AE, Schildberg FW (eds) The immune consequences of trauma, shock and sepsis, Mechanisms and therapeutic approaches. Vol II Pabst Science Publishers, Zagreb, pp 1108–1111Google Scholar
  43. Obayashi T, Yashida M, Mori T, Goto H, Yasuoka A, Iwansaki H, Teshima H, Kohno S, Horiuchi A, Ito A, Yamanguchi H, Shimada K, Kawai T (1995) Plasma (1–3)-ß-d-glucan measurement in diagnosis of deep mycosis and fungal febrile episodes. Lancet 345:17–20PubMedCrossRefGoogle Scholar
  44. Riveau, GR, Novitsky TJ, Roslansky PF, Warren HS, Dinarello CA (1987) Role of interleukin-1 in augmenting serum neutralization of bacterial lipopolysaccharide. J Clin Microbiol 25:889–892PubMedGoogle Scholar
  45. Romero R, Roslansky PF, Oyarzun E, Wan M, Emamian M, Novitsky TJ, Gould MJ, Hobbins JC (1988) Labor and infection. II Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol 158:1044–1049PubMedGoogle Scholar
  46. Saladino R, Garcia C, Thompson C, Hammer B, Parsonnet J, Novitsky TJ, Siber G, Fleisher G (1994) Efficacy of a recombinant endotoxin neutralizing protein in rabbits with Escherichia coli sepsis. Circ Shock 42:104–110PubMedGoogle Scholar
  47. Saladino RA, Fleisher GR, Siber GR, Thompson C, Novitsky TJ (1996a) Therapeutic potential of a recombinant endotoxin-neutralizing protein from Limulus polyphemus. In: Morrison DC, Ryan JL (eds) Novel therapeutic strategies in the treatment of sepsis. Marcel Dekker, New York, pp 97–110Google Scholar
  48. Saladino RA, Stack AM, Thompson C, Sattler F, Novitsky TJ, Siber GR, Fleisher GR (1996b) High-dose recombinant endotoxin neutralizing protein improves survival in rabbits with Escherichia coli sepsis. Crit Care Med 24:1203–1207PubMedCrossRefGoogle Scholar
  49. Siber GR, Saladino RA, Kuppermann N, Nelson D, Parsonnet J, Hammer B, Thompson C, Novitsky TJ, Fleisher GR (1993) Effect of a recombinant endotoxin neutralizing protein from Limulus polyphemus on gram-negative sepsis, In: Levin J, Alving CR, Mumford RS, Stutz P (eds) Bacterial Endotoxin: Recognition and Effector Mechanisms. Excerpta Medica, Amsterdam, pp 379–387Google Scholar
  50. Sloyer JL, Novitsky TJ, Nugent S (2002) Short Communication. Rapid bacterial counts in metal working fluids. J Indust Microbiol Biotechnol 29:323–324CrossRefGoogle Scholar
  51. Stack AM, Saladino RA, Siber GR, Thompson C, Marra MN, Novitsky TJ, Fleisher GR (1997) A comparison of bactericidal/permeability versus increasing protein variant recombinant endotoxin-neutralizing protein for the treatment of Escherichia coli sepsis in rats. Crit Care Med 25:101–105PubMedCrossRefGoogle Scholar
  52. Sullivan JD Jr., Ellis PC, Lee RG, Comb WS Jr., Watson SW (1983) Comparison of the Limulus amoebocyte lysate test with plate counts and chemical analyses for assessment of the quality of lean fish. Appl Environ Microbiol 45:720–722PubMedGoogle Scholar
  53. The Pharmacopoeia of Japan (1992) Bacterial endotoxins test. The Society of Japanese Pharmacopoeia, Tokyo, pp 21–22Google Scholar
  54. Twohy CW, Duran HP, Munson T. (1984) Endotoxin contamination of parenteral drugs and radiopharmaceuticals as determined by the Limulus amebocyte lysate method. J Parenteral Sci Technol 38:190–201Google Scholar
  55. US Department Health and Human Services (1987) Guideline on the validation of the Limulus amebocyte lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devicesGoogle Scholar
  56. US Pharmacopoeia (1995) Bacterial endotoxins test 23. NF 18:1696Google Scholar
  57. Wainwright NR, Miller RJ, Paus E, Novitsky TJ, Fletcher MA, McKenna TM, Williams T (1990) Endotoxin binding and neutralizing activity by a protein from Limulus polyphemus. In: Nowotny A, Spitzer JJ, Ziegler EJ (eds) Cellular and molecular aspects of endotoxin reactions. Elsevier Science Publishers B.V. Amsterdam, pp 315–325Google Scholar
  58. Warren HS, Novitsky TJ, Ketchum PA, Roslansky PF, Kania S, Siber GR (1985) Neutralization of bacterial lipopolysaccharides by human plasma. J Clin Microbiol 22:590–595PubMedGoogle Scholar
  59. Warren HS, Novitsky TJ, Bucklin A, Siber GR (1987a) Endotoxin neutralization with rabbit antisera to Escherichia coli J5 and other gram-negative bacteria. Infect Immun 55:1668–1673PubMedGoogle Scholar
  60. Warren HS, Novitsky TJ, Bucklin A, Kania SA, Siber GR (1987b) Endotoxin neutralization by rabbit antisera to E. coli J5 and other gram-negative bacteria. Infect Immun 55:1668–1673PubMedGoogle Scholar
  61. Warren HS, Novitsky TJ, Martin P, Roslansky PF, Siber GR (1987c) Endotoxin neutralizing capacity of sera from different patient populations assessed by the Limulus lysate test In: Watson SW, Levin J, Novitsky TJ (eds) Detection of bacterial endotoxins with the Limulus amebocyte lysate test. Alan R. Liss, Inc, New York, pp 341–348Google Scholar
  62. Warren HS, Glennon ML, Wainwright NR, Amato SF, Black KM, Kirsch SJ, Riveau GR, Whyte, Zapol WM, Novitsky TJ (1992) Binding and neutralization of endotoxin by Limulus antilipopolysaccharide factor. Infect Immun 60:2506–2513PubMedGoogle Scholar
  63. Watson SW, Novitsky TJ (1991) Horseshoe crabs and marine bacteria. Current Contents 22:10Google Scholar
  64. Watson SW, Novitsky TJ, Quinby HL, Valois FW (1977) Determination of bacterial number and biomass in the marine environment. Appl Environ Microbiol 33:940–946PubMedGoogle Scholar
  65. Weiner DL, Kuppermann N, Saladino RA, Thompson CM, Novitsky TJ, Siber GR, Fleisher GR (1996) Comparison of early and late treatment with a recombinant endotoxin neutralizing protein in a rat model of Escherichia coli sepsis. Crit Care Med 24:1514–1517PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Cape Cod Biosystems, Inc.30 Perch Pond CircleEast FalmouthUSA

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