A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate,,buffer, ahd LÀL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data. The kinetic method had a longer incubation time than the endpoint method and spanned a range of over 3 orders of magnitude compared with the 1-order-of-magnitude rânge of the endpoint assay. The kinetic method was less subject to operator error, since readings were continuous and automatic. The endpoint test was more operator intensive, requiring both addition of acetic atid to stop the reaction and transfer of the sample to thé reading device. A single-step chromogenic reagent was also prepared without lyophilization by mixing reconstituted gel clot LAL with a buffer and a chromogenic substrate. The reagent prepared in this manner performed as well as the colyophilied agent.
A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli 055:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli 055:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.
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