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What is LAL (Limulus Amebocyte Lysate)?

This article was written by Dr. Masakazu Tsuchiya, FUJIFILM Wako Pure Chemical Corporation, for No. 1 (July 1990) of Wako News.
The content of this article is from the time of publication. It is not the latest information due to new knowledge and changes in regulatory rules after original publication.


Ever since Levin and Bang discovered that endotoxin causes the coagulation of Limulus Amebocyte Lysate (LAL)1) , the Limulus test, which applies this phenomenon, has been used in endotoxin detection. The Limulus test has been used recently in a wider variety of fields following improvements to its sensitivity and methodology.

While FUJIFILM Wako sells the LAL reagent, we also conduct research and development studies. Our LAL research team oversees the research on endotoxin manufacturing and Limulus test methodology; thus also double as in-house users of LAL reagents. In addition, we have received inquiries and requests for studies from many users and have gained a lot of experience in the application of the Limulus test. In this series we are providing information about the Limulus test to all users, but we would also like to discuss, as LAL users ourselves, our various experiences and methods.

In this article, for the sake of readers who are new to the Limulus test, we would like to begin by discussing LAL.

1. LAL (Limulus Amebocyte Lysate)

Amoebocytes are cream-colored, spherical blood cells in the horseshoe crab that can move around and spread, like amoebas. These cells can deform and collapse to release intracellular granules when taken out of the body. The cells are collected in a way that keeps them from forming aggregates and when water is added, the extracted cell solution is called LAL.

The coagulation of the blood of horseshoe crabs was already reported by Howell in 18852) . After Levin and Bang discovered the coagulation of LAL by endotoxin in 19641) , the Limulus test rapidly spread as an endotoxin detection method. The reaction mechanism was mainly clarified by Iwanaga et al.3) . According to their research on the LAL coagulation cascade, serine proteases in LAL are sequentially activated by endotoxin molecules. In the last step of the reaction, coagulin forms a gel through the restricted proteolysis of coagulogen. The coagulation reaction is extremely sensitive to low levels of endotoxin contamination; in fact, the gelation of LAL by endotoxin levels in the order of nanograms to picograms has been observed.

The research of Iwanaga et al.3) further revealed the existence of an additional LAL activation pathway through (1→3)-β-D-glucans. Because (1→3)-β-D-glucans are generally found in molds, yeasts and plants, they may readily contaminate LAL test samples and thus the specificity of LAL became a problem. Consequently, there began a demand for endotoxin-specific reagents.

We shall save this issue for another discussion, but there are already endotoxin-specific reagents on the market such as Endospecy (Seikagaku Corporation) and the Limulus-ES test Wako (FUJIFILM Wako Pure Chemical Corporation).

Historically, the rabbit pyrogen test has been the main pyrogen test for drugs. However, as endotoxin is a representative of pyrogens, the Limulus test can be used as a substitution for the rabbit pyrogen test. In the United States there was a movement to adopt the Limulus test as an official compendial method and in 1980 it was finally listed in the US Pharmacopeia XX. In 1988, the Limulus test was also listed in the Japanese Pharmacopeia as the "bacterial endotoxin test."

Although there are currently not many items to which the Limulus test method is applied, it is expected that the Limulus test will replace the Rabbit Pyrogen test for many items in the future. In addition to the testing of drugs and medical devices, the clinical applications of LAL are also being considered.


  1. Levin, J. and Bang, F. B. : Bull. Johns Hopking Hosp., 115, 265 (1964).
  2. Howell, W. H. : Johns Hopking Univ. Circ., 5, 4 (1885).
  3. Nakamura, T. et al. : Japanese Journal of Bacteriology, 88, 781 (1983).

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