First recorded scientific observation of the coagulation of Limulus’ blood.

Frederik B. Bang describes the effects of injecting a marine bacterium into Limulus polyphemus. His results indicate this causes intravascular clotting; other Gram-negative bacteria could cause similar results; the clotting did not require a living bacteria, the bacterial component that caused clotting was not heat labile; and, Gram-positive bacteria did not produce this effect. This finding was the foundation which ultimately lead to the discovery of LAL many years later.

Renewed interest in Limulus polyphemus as a biological model for the study of disease mechanisms.

First detailed modern description of cellular coagulation in Limulus.

Discovery that endotoxin is the key factor in clotting of Limulus blood.

Discovery that bacterial endotoxin (pyrogen) was responsible for the clotting of Limulus blood and that the mechanism was located in the amebocyte granules.

James F. Cooper begins a study under the direction of Jack Levin and Henry N. Wagner to explore the use of LAL as an alternative to using the rabbit pyrogen test to detect endotoxin in pharmaceuticals.

First application of LAL to the diagnosis of human disease.

LAL shown to correlate well with other assays for endotoxin, e.g. Pyrogen (rabbit) Test.

LAL shown it could be applied to the detection of endotoxin in pharmaceutical drugs.

Food and Drug Administration first proposes guidelines for the manufacture of LAL.

Food and Drug Administration proposes standards for the manufacture of LAL. September 18, 1973.

Cooper, Hochstein, and Seligmann Drafted “Return To Sea” Policy.

Travenol Laboratories, Inc. establishes a lysate production laboratory at their Kingstree, South Carolina plant and is using their LAL to test pharmaceuticals both domestically and in some international plants by 1974.

Beginning with Associates of Cape Cod, Inc., Woods Hole, MA, in September 1977, by October 1978 there were three additional LAL manufacturers licensed by the FDA. These were, in order of licensing: Mallinckrodt Inc., St. Louis, MO, Microbiological Associates, Walkersville, MD, and Travenol Laboratories, Morton Grove, IL

FDA allows substitution of LAL for the official rabbit pyrogen test when testing biological products and medical devices providing approval is first obtained from the appropriate bureau of the FDA.

FDA proposal for the live release of horseshoe crabs back to their native environment after only one blood collection.  (This regulation was rescinded in 1996 under the Federal Reinvention of Government (REGO) Initiative.)

Attempt at Limulus aquaculture.

Worthington Laboratories, Freehold, NJ joins the group of laboratories manufacturing LAL (Pyrostat^TM brand).

Warning that: “The emerging importance of Limulus to biomedical research, as the source of Limulus lysate, requires more complete knowledge of the biology of the species, so that it can be wisely managed as a natural resource.”

Warning that biomedical use as well as use of Limulus for conch bait in addition to habitat destruction requires preparation of a practical plan for the conservation of Limulus.

Attempt to grow Limulus’ amebocytes in culture (for the in vitro production of LAL).

Review of Limulus and other invertebrate clotting with emphasis on innate infection defense mechanisms.

In the FEDERAL REGISTER of January 18, 1980 (45 FR 3668), FDA announced the availability of a draft guideline that set forth procedures for use of the LAL test as an end-product testing method for endotoxins in human and animal injectable drug products. This draft guideline was made available to interested parties to permit manufacturers, especially those who had used the LAL test in parallel with the rabbit pyrogen test, to submit data that could be considered in the preparation of any final guideline.

The United States Pharmacopeial Convention (USP) publishes General Chapter <85> Bacterial Endotoxins Test in USP XX, making the LAL test a Compendial method to detect bacterial endotoxin in pharmaceutical products and medical devices.

The United States Food and Drug Administration publishes Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, describing FDA’s opinion regarding the appropriate methods for validation and use of LAL for detecting the presence of endotoxin in medical products.

Dr. James and Frances Cooper wrote State legislation for approval in 1991 that provided for the management and regulation of the horseshoe crab fishery, which resulted in a horseshoe crab harvesting ban for all but biomedical use.

LAL methodologies advanced from gel clot and turbidimetric to the colorimetric techniques of endpoint and kinetic chromogenic in the late 1980s and early 1990s.

The Atlantic States Marine Fisheries Commission (ASMFC) establishes a formal fisheries management plan for horseshoe crab populations’ coast wide.

Jay Harrington (horseshoe crab fisherman) sues the US Department of Interior on behalf of Associates of Cape Cod, Inc. to continue to allow the harvest by special permit of horseshoe crabs in the Monomoy NWR (Cape Cod). An injunction is issued to his benefit allowing continued harvest for two seasons but final ruling closes harvest of horseshoe crabs in the NWR permanently. Although harvest pressure is moved to other areas of Cape Cod and Southeastern MA, horseshoe crab numbers today (2013) are higher than any other area in New England.

The Cape Cod (MA) National Seashore closes waters in its boundaries to the harvest of horseshoe crabs. Although other fishing activities in the National Park are allowed by charter, e.g. shellfishing, the Park designates the horseshoe crab as “wildlife” and not a “shellfish” thus extending its protection (even for biomedical use and with conditions that bled crabs be returned to the area) in the Park. The largest area affected is a part of Pleasant Bay, MA, another Harrington fishing location.

Carl N. Shuster Sanctuary established (no harvesting of horseshoe crabs for any reason within the sanctuary boundaries).

There are four (4) companies licensed by the FDA in the United States to manufacture and market LAL commercially. These were (with their accompanying areas of horseshoe crab harvest):

• Associates of Cape Cod, Inc., Woods Hole, MA (Cape Cod, Southeastern Massachusetts, Rhode Island)

• Mallinckrodt Inc., St. Louis, MO (Various locations in North Carolina)

• BioWhittaker (originally Microbiological Associates, acquired by Cambrex in 1997) (Chincoteague, VA)

• Haemachem, St. Louis, MO (Various locations in North Carolina and Long Island, NY)

• Charles River Endosafe (originally Endosafe, Inc. acquired by Charles River Laboratories in 1995) (Various locations in South Carolina and contract processed blood from Cape May, New Jersey via Limuli Laboratories, Inc., an outgrowth of Marine Biologicals Inc., at one time licensed by the FDA to produce a final LAL product.)

Baxter Travenol, Deer Lake, IL, still retains a license to manufacture LAL and use the product in their own facility but do not market LAL commercially.

An alternative method for endotoxin detection that used a recombinant form of Factor C (rFC) from the horseshoe crab was introduced by the National University of Singapore.

Cambrex BioScience (formerly BioWhittaker) licenses the rFC technology from the National University of Singapore, and commercializes their rFC product PyroGene™.

The Endosafe®-PTS™ for endotoxin testing is licensed by the FDA after three years of use in R&D application. Conforming to the FDA initiative for process analytical technology (PAT), the portable incubating spectrophotometer provides rapid endotoxin testing results at the point of sample collection, contributing to improved quality, safety and efficiency in the manufacturing process.

Charles River launched the Endosafe®-PTS™ aboard the International Space Station, where astronauts began using the system to perform biological studies necessary for an extended human presence in space–from crew health and spacecraft environmental studies to the search for life elsewhere in the solar system. NASA used the Endosafe®-PTS™ to monitor the environment for microbial contamination during the construction of the Mars exploration rovers “Spirit” and “Opportunity”. Future applications of PTS may include monitoring the spread of Earth-derived biological material to the Moon and detecting signs of microbial life on Mars.

In July, FDA withdraws the 1987 LAL Guidance document, stating that it was obsolete and would be replaced in the future.

EndoLISA, innovative ELISA-test using an Endotoxin-specific phage protein for binding and rFC for detecting endotoxin is introduced by Hyglos GmbH of Munich, Germany

In June, FDA issues the less-prescriptive Guidance for Industry – Pyrogen and Endotoxins Testing: Questions and Answers as a replacement to the 1987 Guidance document.  The new document states the use of recombinant Factor C methods is accepted by FDA if validated as per USP General Chapter <1225> Validation of Compendial Methods.

In January, Hyglos launches their recombinant Factor C (rFC) endotoxin detection assay EndoZyme®.

There are still four companies licensed by the FDA to produce LAL in the US:

• Associates of Cape Cod, Inc., Falmouth, MA now wholly owned by the Tokyo-based Seikagaku Corporation

• Charles River Endosafe, Charleston, SC

• Lonza, a Swiss-based company with LAL facilities in Walkersville, MD (formerly BioWhittaker/Cambrex, also includes original technology from Mallinckrodt, Inc. no longer in the business themselves)

• WAKO Pure Chemical Company, an Osaka-based company that acquired Haemachem, Inc. with a US base in Richmond, VA and an LAL facility on the VA shore.

In August, Hyglos introduces the Endo-RS, Endotoxin Recovery Kit, a sample preparation method for biopharmaceutical formulations affected by the Low Endotoxin Recovery (LER) phenomenon.

In July, the European Pharmacopoeia publishes the revised Chapter 5.1.10, Supplement 8.8, including Recombinant Factor C (rFC) as an alternative method since, “this practice avoids the use of animal species“.

“The use of alternative reagents such as recombinant factor C as a replacement to the amoebocyte lysate eliminates the use of a reagent extracted from live animals. Replacement of a rabbit pyrogen test or a bacterial endotoxin test prescribed in a monograph by a test using recombinant factor C reagent or any other reagent as a replacement of the amoebocyte lysate is to be regarded as the use of an alternative method in the replacement of a pharmacopoeial test, as described in the General Notices.”

Kikuchi et al. of PMDA, part of the Japanese Pharmacopeia, publish a comparison study of three LAL tests and three rFC tests showing equivalence.

Hyglos/bioMérieux introduce ENDOZYME II GO, rFC endotoxin testing made significantly easier and faster.

Fujifilm Wako Chemicals U.S.A. Corporation successfully became an active participant in the FDA collaborative study to assess the sensitivity of LAL reagent candidate material for the gel-clot assay

FDA approves first drug using the recombinant Factor C (rFC) Assay for endotoxin testing of Eli Lilly’s Emgality™ , the first drug approved by the U.S. Food and Drug Administration (FDA) to have been released using this method instead of traditional Limulus Amebocyte Lysate (LAL) – based methods. Emgality™ is a monoclonal antibody drug treatment for the prevention of migraine in adults.

Associates of Cape Cod, Inc. introduces PyroSmart NextGen™, the first commercially available sustainable recombinant Cascade Reagent (rCR). ACC’s PyroSmart NextGen™ uses the same cascade as traditional LAL reagents by manufacturing the Factors responsible for the cascade using recombinant processes.