Automation of the Limulus amoebocyte lysate test by using the Abbott MS-2 microbiology system

Jorgensen, James H.; Alexander, G A

doi:10.1128/aem.41.6.1316-1320.1981

Cited by 20 publications

(3 citation statements)

References 23 publications

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“…All previously described endpoint chromogenic methods, as well as endpoint tùrbidimetric methods (15), have ranges of detection limited to little more than 1 order of magnitude. By contrast, the kinetic turbidimetric method successfully used the kinetics of endotoxin activation of the LAL enzymatic cascade (measured as turbidity) to increase the range of detection to 5 brders of magnitude (6,7,9,12). This report describes both an endpoint method and a kinetic chromogenic LAL method which use a single reagent mixture.…”

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confidence: 99%

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin

1989

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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.

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confidence: 99%

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin

1989

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“…Derivation of the rate of the development of the turbidity reaction is complicated by the sigmoid shape of the reaction curve, and hence some form of mathematical transformation of the OD readings is required (73,166,351,352,385). Several less complicated approaches to estimating the rate of the reaction, such as the time to reach a given ''threshold'' OD reading (81,185,215), time to gelation (57), or the time taken between two predetermined OD readings (153), have been described.…”

Section: Optimal Conditions For Lal Reactionmentioning

confidence: 99%

Endotoxemia: methods of detection and clinical correlates.

Hurley¹

1995

Clinical Microbiology Reviews

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“…The first step toward automation of the LAL test was the development and application of turbidimetric and chromogenic assays that allowed the use of spectrometers for the simultaneous readout of more than one sample and thus provided significant improvements compared to the labor-intensive gel clot method . There was also some study in the 1980s to automate these assays using robotic pipetting systems .…”

Section: Introductionmentioning

confidence: 99%

Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics

et al. 2021

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We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 μL/reaction to 9.6 μL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.

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Automation of the Limulus amoebocyte lysate test by using the Abbott MS-2 microbiology system

Cited by 20 publications

References 23 publications

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin

Endotoxemia: methods of detection and clinical correlates.

Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics

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